Proline-based neuropeptide FF receptor modulators

ABSTRACT

Neuropeptide FF receptor modulators based on a proline scaffold are provided which offer NPFF receptor potencies in the nanomolar range and antagonistic selectivity for the NPFF1 receptor. Methods, compounds and compositions for modulating the function of neuropeptide FF receptors are provided for pharmacotherapies capable of influencing conditions or disorders affected by the neuropeptide FF receptors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional under 35 USC § 120 of U.S. patent application Ser.No. 16/485,570 filed Aug. 13, 2019 in the names of Yanan Zhang and ThuyNguyen for “PROLINE-BASED NEUROPEPTIDE FF RECEPTOR MODULATORS”, which inturn is a U.S. national phase under 35 USC § 371 of International PatentApplication No. PCT/US18/18074 filed Feb. 13, 2018 in the names of YananZhang and Thuy Nguyen for “PROLINE-BASED NEUROPEPTIDE FF RECEPTORMODULATORS”, which in turn claims the benefit under 35 USC § 119 of U.S.Provisional Patent Application No. 62/458,731 filed Feb. 14, 2017 in thenames of Yanan Zhang and Thuy Nguyen for “PROLINE-BASED NEUROPEPTIDE FFRECEPTOR MODULATORS”. The disclosures of all such applications arehereby incorporated herein by reference, in their respective entireties,for all purposes.

FIELD

The present disclosure relates to proline-based compounds specific forneuropeptide FF (NPFF) receptors. The present disclosure further relatesto methods, compounds and compositions for modulating the function ofneuropeptide FF receptors to provide pharmacotherapies capable ofinfluencing conditions or disorders affected by the neuropeptide FFreceptors.

DESCRIPTION OF THE RELATED ART

Neuropeptide FF (NPFF) belongs to a family of neuropeptides calledRFamide peptides, members of which all contain an Arg-Phe-NH₂ (RF-amide)motif at their C terminus. Neuropeptide FF is an endogenous peptide thatbinds to and activates two G protein-coupled receptors (GPCR), NPFF1(GPR147) and NPFF2 (GPR74). These receptors are members of the rhodopsinfamily and predominantly couple to the Gα_(i/o) proteins. Originallyisolated from bovine brain, NPFF and its receptors have been identifiedin the central nervous system (CNS) of various animal species. Ligandbinding studies performed on rodents confirmed that both receptorsubtypes are widely expressed in the brain, whereas only NPFF2 receptorsare expressed in the spine at detectable levels. The NPFF system hasbeen implicated in the regulation of a variety of physiologicalprocesses, such as insulin release, food intake, memory, blood pressure,electrolyte balance, and neural regeneration. The NPFF system has alsobeen shown to play an important role in modulating the effects ofopioids and several other classes of drugs of abuse.

It is well documented that NPFF, having no affinity for the opioidreceptors, is a modulator of opioid receptor function and attenuates thetolerance and dependence to opioids. Several studies have shown that theeffects of NPFF on opioid modulation are dependent on the route ofadministration. For example, intracerebroventricular (i.c.v.)administration of NPFF in rats attenuated morphine-induced analgesia andlocomotion, and precipitated opioid withdrawal syndromes inmorphine-dependent rats, whereas intrathecal (i.t.) administrationproduced opioid-induced analgesia and also prolonged morphine-inducedanalgesia (Malin et al., Peptides, 11, 277-280 (1990); Yang et al., ProcNatl Acad Sci USA, 82, 7757-61, 1985; Gouarderes et al., Eur. J.Pharmacol., 237, 73-81 (1993)). Injection (i.c.v.) of 1Dme, a peptidicNPFF analog, inhibited morphine induced analgesia as well as theacquisition of place conditioning by morphine (Marchand et al.,Peptides, 27, 964-72, 2006). Ventricular injection of NPFF antiserumrestored the analgesic response to morphine in morphine-tolerant ratsbut did not affect opiate-naïve rats (Lake et al., Neurosci. Lett.,132(1):29-32, 1991). RF9, a dipeptide NPFF1/2 receptor antagonist,dose-dependently blocked the long-lasting hyperalgesia produced byeither acute fentanyl or chronic morphine administration (Elhabazi etal., Br. J. Pharmacol., 165, 424-435, 2012; Simonin et al., Proc. Natl.Acad. Sci. USA, 103, 466-471, 2006). This effect appears to be mainlymediated by the NPFF1 receptor, as the selective NPFF1 antagonistAC-262620 also reduced opioid tolerance (Lameh et al., J. Pharmacol.Exp. Ther., 334, 244-254, 2010).

Opioids remain the most effective analgesics for many pain conditions,particularly for chronic pain; however, the adverse effects related toopioid use such as physical dependence, hyperalgesia and tolerancepreclude adequate dosing and effective pain control in a largepopulation of pain sufferers. Combination therapy which combines opioidswith other drugs that may increase the efficacy of opioids and/or reducethe untoward effects offers a promising alternative strategy for painmanagement.

In view of the biological activity believed to be modulated by NPFF, theart is seeking compounds and compositions which provide inhibition oractivation of the functional effects of NPFF.

SUMMARY

The present disclosure relates to neuropeptide FF and the discovery ofproline-based neuropeptide FF receptor modulators. In one aspect, thedisclosure relates to such proline-based neuropeptide FF receptormodulators according to Formula (I):

wherein R₂ is selected from —N—(C₂-C₈alkyl)₂ and NH—R₁, wherein R₁ isselected from C₂-C₉ alkyl, heterocyclealkyl, cycloalkylalkyl,aminoalkyl, and arylalkyl; R₃ is selected from C₃-C₉ alkyl, aryl,heteroaryl, heterocycle, heteroarylalkyl, heterocyclealkyl, andarylalkyl; R₄ is selected from H and C₁-C₂ alkyl; and R₅ is selectedfrom C₃-C₉ alkyl, heteroarylalkyl, heteroaryl, heterocyclealkyl,heterocycle, cycloalkylalkyl, and arylalkyl. Proline-based neuropeptideFF receptor modulators according to Formulas II, IIA, III and IV arealso provided.

In another aspect, the disclosure relates to a pharmaceuticalcomposition comprising a proline-based neuropeptide FF receptormodulator represented by Formulas I, II, IIA, III or IV or apharmaceutically acceptable salt thereof and a pharmaceuticallyacceptable carrier.

In a further aspect, the disclosure relates to a method for treating asubject having or susceptible to a condition or disorder wheremodulation of neuropeptide FF receptor activity is of therapeuticbenefit, comprising administering to said subject having or susceptibleto said condition or disorder a therapeutically effective amount of acompound according to Formulas I, II, IIA, III or IV or apharmaceutically acceptable salt thereof.

Other aspects, features and embodiments of the disclosure will be morefully apparent from the ensuing description and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the chemical structures of various NPFF ligands describedin the art.

FIG. 2 depicts the chemical structure of proline-based compound 1.

FIG. 3 is a graphical representation of NPFF EC₅₀ in calciummobilization assays, showing data obtained when stable NPFF1 and NPFF2cell lines were treated with NPFF.

FIG. 4 shows concentration-response curves of compound 16 in the NPFF1and NPFF2 calcium mobilization assays, with graph A showing theantagonist activity of compound 16 in the NPFF1 calcium mobilizationfunctional K_(e) assay, for NPFF alone (∘) and NPFF+5 μM final 16 (□) instable NPFF1-RD-HGA16 cells, and with graph B showing the antagonistactivity of compound 16 in the NPFF2 calcium mobilization functionalK_(e) assay, for NPFF alone (∘) and NPFF+10 μM final 16 (□) in stableNPFF2-RD-HGA16 cells.

FIG. 5 shows concentration-response curves of compounds 16 and 33 in theNPFF1 and NPFF2 cAMP assays, with graph A showing the antagonistactivity of compounds 16 and 33 in the NPFF1 cAMP functional K_(e)assay, for NPFF alone (∘), NPFF+4 μM final 16 (□), and NPFF+2 μM final33 (⋄) in stable NPFF1-CHO cells, and with graph B showing theantagonist activity of compounds 16 and 33 in the NPFF2 cAMP functionalK_(e) assay, for NPFF alone (∘), NPFF+10 μM final 16 (□), and NPFF+10 μMfinal 33 (⋄) in stable NPFF2-CHO cells.

FIG. 6 shows the results of testing in a fentanyl-induced hyperalgesiamodel in rats, in which graph A shows results for fentanyl-inducedmechanical hyperalgesia, and in which graph B shows theanti-hyperalgesic effects of compounds 16 and 33 (N=6 per group).

DETAILED DESCRIPTION

The present disclosure relates to proline-based neuropeptide FF receptormodulators. The modulators exhibit affinity for and activity at theneuropeptide FF receptors. The molecules may thus be useful in thetreatment of disorders, syndromes and conditions mediated by modulationof the neuropeptide FF receptors.

Research has found that NPFF precipitates a nicotine withdrawalsyndrome, also suggesting that NPFF participates in the processes ofdependence and drug addiction. (Malin et al., Pharmacol. Biochem.Behav., 54, 581-585, 1996) It has further been shown that chronicadministration of NPFF into the lateral ventricle potentiated thebehavioral sensitization to amphetamine. (Chen et al., Brain Res., 816,220-224, 1999) More recently, it was demonstrated that stimulation ofNPFF receptors decreased the expression of amphetamine-inducedcondition-placed preference, while the inhibition of NPFF receptorsdecreased amphetamine withdrawal anxiety. (Kotlinska et al., Peptides,33, 156-163, 2012) Moreover, it has been suggested that NPFF is involvedin the mechanism of expression of sensitization to cocainehyperlocomotion, although this effect could be non-specific. (Kotlinskaet al., Peptides, 29, 933-939, 2008) Consistent with these observations,there appears to be evidence that NPFF binding sites are abundant in theventral tegmental area (VTA), while NPFF-like immunoreactivity wasdetected in the nucleus accumbens (NAc), two brain regions belonging tothe mesolimbic dopamine (DA) projections which are known to be involvedin drug addiction. (Wu et al., Peptides, 31, 1374-1382, 2010) Together,these findings make the NPFF system appear to be a viable target for thetreatment of drug addiction.

In addition to NPFF, several other neuropeptides from the RFamide familyhave been found to activate one or both NPFF receptors, including NPSF(neuropeptide SF), NPAF (neuropeptide AF) and NPVF (neuropeptide VF). Anumber of peptidomimetic NPFF ligands have been reported, which includethe guanidine functional group (FIG. 1 ). In these ligands, acylation ofthe last two amino acid residues (RF) have been reported to be criticalfor NPFF activities (BIBP3226 (Bonini et al., J. Biol. Chem.275(50):39324-39331 (2000), chemical structure 1, FIG. 1 ) and RF9(Simonin et al., PNAS 103(2): 466-471 (2006), chemical structure 2, FIG.1 ). Modification of RF9 led to the NPFF1 selective dipeptidebiphenyl-RF and peptidomimetic RF313 (Bihel et al., ACS Chem Neurosci.,6(3): 438-445 (2015), chemical structure 3, FIG. 1 ; Gealageas, et al.,Bioorg. Med. Chem. Lett. 22, 7471-7474 (2012); Elhabazi, et al.,Neuropharmacol., 118, 188-198 (2017)). These peptides or peptidomimeticswere found to be effective in preventing fentanyl-induced hyperalgesiain rats by subcutaneous or oral administration, acting as antagonists.

Several classes of non-peptide NPFF ligands have also been disclosed.Quinazolino-, pyrimidine-, thiazole- and quinolino-guanidines werereported as NPFF ligands (WO 03/026667, chemical structure 5, FIG. 1 ;U.S. Pat. No. 7,544,691, chemical structures 4, FIG. 1 ). A series ofN-benzylpiperidines were found to have mixed activities asagonists/antagonists at NPFF1 and antagonists at NPFF2 (Journigan et al,J. Med. Chem., 57, 8903-27, 2014, chemical structure 6, FIG. 1 ). Theseseries have the guanidine functionality. While the guanidinefunctionality has been in some cases associated with high plasma-proteinbinding and limited BBB penetration, some of these guanidine-containingligands have been reported to enter the CNS, although to a relativelysmall extent. So far, only two series of small molecule NPFF ligandsthat do not possess the guanidine functionality have been reported, butthe in vivo effects of these ligands are yet to be investigated (WO2009/038012, chemical structure 7, FIG. 1 ; WO 2004/080965, chemicalstructure 8, FIG. 1 ).

Small non-peptidic compounds are not subject to peptidolytic degradationand thus are more favorable tools to explore biological roles of NPFFreceptors. In an effort to develop novel small molecule NPFF ligands, ahigh throughput screen of a GPCR-oriented compound library wasconducted. Compound 1 with a proline scaffold, shown in FIG. 2 , emergedas a promising lead with moderate activities on two NPFF subtypes withreasonable physiochemical properties. A synthetic route to this scaffoldwas developed and a focused library of proline analogs was prepared inorder to explore the structure-activity relationships (SARs) at threeregions of this scaffold (FIG. 2 ).

The initial SAR investigation focused on three regions, the carboxamide,the amino center and the 4-position of the proline and revealedsubstitution at these positions influenced the NPFF antagonism andsubtype selectivity. A number of compounds with submicromolar NPFF1potency have been identified. For example, compound 16 with an n-pentylamino functionality had a K_(e) value of 720 nM at NPFF1 and >4 foldpreference over NPFF2. Compound 33 with a 4-nitrophenethyl substituentemerged as the most potent analog at NPFF1 (K_(e)=245 nM) and ˜3 foldpreference over NPFF2. In general, these compounds were more potent atthe NPFF1 receptor, but selectivity was only modest against NPFF2.Results from the secondary cAMP assay further confirmed the NPFFantagonistic activities of these compounds and radioligand bindingassays demonstrated that the ligands bind to the NPFF receptors withmoderate affinity.

The representative compounds 16 and 33 possess moderate solubility andblood-brain barrier permeability, demonstrating the proline scaffold asa potential druglike and potent NPFF template.

The compounds obtained were characterized in calcium mobilization assaysto evaluate their activities at both NPFF receptors. Several compoundswere further evaluated for their effect in modulating cellular levels ofcyclic adenosine monophosphate (cAMP) and their binding affinity to thetwo NPFF receptors. The drug-like properties such as solubility andblood-brain barrier permeability were then examined. Finally, theeffects of these compounds in reversing fentanyl-induced hyperalgesiawere investigated.

Traditionally, NPFF activity has been examined using assays such asradiolabeled (radioligand) binding, GTP-γ-S or cAMP assay. To establisha platform allowing for low-cost high-throughput screening, calciummobilization assays were developed using Chinese hamster ovary (CHO)cells simultaneously over expressing Gα₁₆ protein and either human NPFF1or NPFF2 receptors. The NPFF1 and NPFF2 stable cell lines were createdby transfecting the expression plasmids into RD-HGA16 CHO cells(Molecular Devices), selecting for positive clones using antibioticresistance, and testing for functional expression of NPFF1 and NPFF2receptors following procedures previously disclosed (Zhang, Y. et al., JMed Chem, 53, 7048-60, 2010; German, N. et al., J Med Chem, 57, 7758-69,2014; Nguyen, T. et al., Bioorg Med Chem, 23, 2195-203, 2015). Cloneswere first screened against 10 μM NPFF to identify clones that hadfunctional NPFF receptors. Eight clones with the highest maximal NPFFresponse were further evaluated with NPFF concentration-response curves;the clone with the most potent and efficacious NPFF response was chosenas the working clone.

FIG. 3 shows the data obtained when the stable NPFF1 and NPFF2 celllines were treated with NPFF. In the NPFF1-RD-HGA16 cells, NPFF has anEC₅₀ value of ˜62 nM and the signal window is 15,000 relativefluorescent units (RFUs). In the NPFF2-RD-HGA16 cells, NPFF has an EC₅₀value of ˜22 nM and the signal window is 8,200 RFUs. These NPFF EC₅₀values are consistent with results from other cAMP and GTP-γ-S assays(Gouarderes, C. et al., Neuropharmacology, 52, 376-86, 2007; Lameh, J.et al., J Pharmacol. Exp. Ther., 334, 244-54, 2010; Vyas, N. et al.,Peptides, 27, 990-60, 2006).

In parental RD-HGA-16 CHO cells, there was no response to NPFF,confirming its signaling through NPFF receptors. The NPFF1 stable cellline was successfully miniaturized from 96- to 384-wells (Z′factor=0.75) for library screening.

The NPFF1 FLIPR-based (fluorometric imaging plate reader) calciummobilization assay was used for the high throughput screen for ligandsfrom an in house GPCR-enriched library as well as to characterize thesynthetic compounds obtained. The screened library was highly diverse,including 22,000 compounds, and its content and properties werecritically appraised for screening against GPCRs based on a number offactors including maximum diversity, optimal ADME parameters, structuralnovelty (minimal overlap with scaffolds found in known GPCR ligands anddrugs), and pharmacophoric compliance with characteristics prototypicalof GPCR ligands.

Library compounds were screened at 10 μM final concentration for bothagonist and antagonist activities as part of a 3-addition protocol thatwas developed, enabling evaluation of both modes of activity with asingle assay plate, thereby reducing the overall time and cost of thescreening. Concentration-response curves were run with 37 selectedantagonists (27 with >65% inhibition and 10 with >85% inhibition)resulting in the confirmed activities of these compounds.

Thus, all of the synthesized compounds were first characterized in NPFF1and NPFF2 calcium mobilization assays for their ability to antagonizeNPFF stimulated calcium influx. Since the NPFF receptors natively coupleto Gα_(i/o) proteins and inhibit adenylate cyclase, active compoundswith apparent dissociation constant K_(e) values of ≤1 μM in NPFF1 orNPFF2 calcium assays were selected, and further evaluated inPerkinElmer's Lance cAMP assays.

In both functional assays, EC₅₀ curves of the agonist NPFF were obtainedalone and together with the test compound, and the right-shift of theagonist curve was measured. The apparent dissociation constant K_(e) wascalculated from compound-mediated inhibition of NPFF activity aspreviously described (Perrey et al., J. Med. Chem., 56, 6901-16, 2013;Perrey et al., ACS Chem. Neurosci. 6, 599-614, 2015). As indicatedabove, all compounds were tested for agonist activity using the calciummobilization assay; none showed any significant agonist activity at theeither the NPFF1 or NPFF2 receptors (<20% of NPFF E_(max) at 10 μMfinal). These compounds were also characterized in a radioligand bindingassay to measure affinity and confirm that the proline scaffold is abona fide template as an NPFF ligand. Kinetic solubility andbidirectional MDCK-MDR1 permeability assays of the compounds wereperformed by Paraza Pharma Inc. (Montreal, Canada) according to theirstandard protocols.

As a result of the above-described research, proline-based NPFF receptormodulators were discovered according to Formula I.

wherein R₂ is selected from —N—(C₂-C₈alkyl)₂ and NH—R₁, wherein R₁ isselected from C₂-C₉ alkyl, heterocyclealkyl, cycloalkylalkyl,aminoalkyl, and arylalkyl; R₃ is selected from C₃-C₉ alkyl, aryl,heteroaryl, heterocycle, heteroarylalkyl, heterocyclealkyl, andarylalkyl; R₄ is selected from H and C₁-C₂ alkyl; and R₅ is selectedfrom C₃-C₉ alkyl, heteroarylalkyl, heteroaryl, heterocyclealkyl,heterocycle, cycloalkylalkyl, and arylalkyl; or a pharmaceuticallyacceptable salt, amide, ester or prodrug thereof.

Formula I represents proline-based NPFF modulators where each of thethree regions of the proline scaffold of Compound 1 may be optimized.

In embodiments of Formula I, wherein R₁ is selected fromheterocyclealkyl, cycloalkylalkyl, aminoalkyl, and arylalkyl; R₃ isselected from heteroarylalkyl, heterocyclealkyl, and arylalkyl; and/orR₅ is selected from heteroarylalkyl, heterocyclealkyl, cycloalkylalkyl,and arylalkyl, the alkyl group is C₁, C₂ or C₃. Suitable examples ofsuch groups are shown in the Tables below.

In certain embodiments of Formula I, R₂ is —N—(C₅-C₆alkyl)₂.

In certain embodiments of Formula I, R₂ is NH—R₁. In further embodimentsof Formula I, when R₂ is NH—R₁, R₁ is C₃-C₆ alkyl.

In some other embodiments of Formula I, when R₂ is NH—R₁, R₁ is benzylor phenethyl, substituted or unsubstituted. In certain embodiments whereR₁ is substituted phenethyl, the phenethyl is substituted by loweralkoxy such as methoxy, nitro, lower alkyl, halogen, or halogenatedlower alkyl such as CF₃. The phenethyl group may be monosubstituted ordisubstituted.

Embodiments of R₂ as described herein may be combined with anycombination of embodiments of R₃, R₄ and R₅ described herein.

In certain embodiments of Formula I, R₃ is substituted benzyl orsubstituted phenethyl with a diversity of substituents. By way ofexample, the substituents may be halogen, methoxy, methyl, cyano, N—CH₂,among others.

In certain embodiments of Formula I, R₃ is benzyl or substituted benzyland R₄ is H. In such embodiments where R₃ is substituted benzyl, thebenzyl is mono- or di-substituted with methoxy.

In certain other embodiments of Formula I, R₃ is benzyl or substitutedbenzyl and R₄ is methyl.

In embodiments of Formula I where R₃ is monosubstituted benzyl orphenethyl, the substituent is at the 4-position.

In some embodiments of Formula I, R₅ is benzyl or substituted benzyl. Insuch embodiments where R₅ is substituted benzyl, the substituents may behalogen or methoxy. In further embodiments where R₅ is substitutedbenzyl, the benzyl is monosubstituted and the substituents may beortho-substituted halogen or methoxy.

In one embodiment, the proline-based NPFF receptor modulators may berepresented by Formula II:

wherein R₂ is selected from —N—(C₂-C₈alkyl)₂ and NH—R₁, wherein R₁ isselected from C₂-C₉ alkyl, heterocyclealkyl, cycloalkylalkyl,aminoalkyl, and arylalkyl; and X is S, SO, SO₂, O, NH or CH₂.Proline-based NPFF modulators represented by Formula II havemodifications in Region 1, the carboxamide region, of Compound 1.

In certain embodiments of Formula II, R₂ is —N—(C₅-C₆alkyl)₂.

In certain embodiments of Formula II, X is S or O.

According to some embodiments of Formula II, R₂ is NH—R₁, wherein R₁ isselected from C₂-C₉ alkyl, heterocyclealkyl, cycloalkylalkyl,aminoalkyl, and arylalkyl; and X is S, SO, SO₂, O, NH or CH₂. Thisembodiment is represented by Formula IIA:

In certain embodiments of Formula IIA, R₁ is C₃-C₆ alkyl; and X isoxygen. In other embodiments of Formula IIA, R₁ is benzyl or phenethyl,substituted or unsubstituted and X is oxygen. In certain embodimentswhere R₁ is substituted phenethyl, the phenethyl is substituted by loweralkoxy, such as methoxy, nitro, lower alkyl, halogen, or halogenatedlower alkyl such as CF₃.

Table 1 lists the SAR, determined as discussed above, of embodimentscorresponding to Formula II wherein X is oxygen.

TABLE 1

Compound R₂ NPFF1 Ke (nM)^(a) NPFF2 Ke (nM)^(a) 10 NHMe >10,000^(b)>10,000^(b) 11 NHEt >10,000^(b) >10,000^(b) 12 NH(n-Pr) 2,600 ± 1509,080 ± 220^(b) 13 NH(n-Bu) 1,240 ± 140 d 14 NH(s-Bu) 1,500 ± 60  3,680± 350^(b) 15 NH(t-Bu) 1,880 ± 210 1,260 ± 80^(c)  16 NH(n-Pentyl)  720 ±10 3,090 ± 580^(b) 17 NH(i-Pentyl) 1,320 ± 90  1,540 ± 130^(c) 18NH(n-hexyl)  820 ± 80 1,490 ± 200^(c) 19 NH(n-decyl) >10,000^(b,c) d 20

1,960 ± 70  d 21

1,120 ± 230 1,220 ± 360^(c) 22

5,460 ± 490^(b)   d 23

 780 ± 80^(c) 2,010 ± 440^(c) 24

  850 ± 140 >10,000^(b) 25

>10,000 2,600 ± 280^(c) 26

 670 ± 60 1,750 ± 110^(c) 27

1,030 ± 50  d 28

1,200 ± 250 2,080 ± 380^(d) 29

2,510 ± 80  >10,000^(b) 30

7,500 ± 1,850^(b) >10,000^(b) 31

e 2,420 ± 260^(c) 32

  990 ± 220^(c) 1,830 ± 90^(c)  33

 250 ± 60^(c)   690 ± 140^(c) 34

  610 ± 130 3,490 ± l,370^(b,c) 35

1,130 ± 110 9,880 ± 180^(b) 36

1,670 ± 220 d 37

1,820 ± 200 d 38

>10,000^(b) >10,000^(b) 39

1,880 ± 250 d 40 NEt₂ 2,900 ± 150 4,930 ± 20^(b)  41 N(n-Pr)₂   880 ±120^(c) 1,730 ± 270^(c) 42

1,330 ± 290^(c) 2,080 ± 300^(c) ^(a)Values are the mean ± SEM of atleast three independent experiments in duplicate. ^(b)Values are themean ± SME of two independent experiments in duplicate.^(c)Pre-incubation of antagonist and test compound was 45 min or 1 hr.^(d)Compound was inactive in antagonist screen at 10 μM final (N = 2).^(e)Compound appeared cytotoxic in the assay and potency was notdetermined.

Analogs with a 4-methoxybenzyl group at the 4-position of the prolinecore, instead of the 4-(methylthio)benzyl group in compound 1, were usedin the SAR studies at the carboxamide region because the methoxy moietyis known to have better metabolic stability. Moreover, the correspondingstarting materials for the methoxy analogs are more readily availablecompared to the 4-(methylthio)benzaldehyde.

As can be seen from Table 1, the NPFF1 antagonist activity of thisseries was sensitive to the length of the R₁ substituent of the amidefunctionality. Methyl and ethyl analogs (10 and 11) were inactive at 10μM at both NPFF receptors. The antagonist activities at the NPFF1receptor increased from n-propyl to n-pentyl (12, 13, 16), thendecreased slightly with n-hexyl (18) and was completely abolished withn-decyl chain (19). Among the three butyl isomers (13-15), the NPFF1antagonist activity slightly decreased in the order of n-butyl, s-butyland t-butyl. In contrast, the NPFF2 antagonist activity increasedsignificantly in the same order, indicating that a linear chain ispreferred for NPFF1 selectivity. A similar trend was observed withn-pentyl and iso-pentyl isomers (16 and 17). Between these two isomers,n-pentyl is the more potent and selective NPFF1 antagonist (NPFF1K_(e)=720 nM, NPFF2 K_(e)=3,090 nM).

When the cyclohexyl (21) was replaced by a phenyl group (24), the NPFF1activity was slightly improved while retaining selectivity over theNPFF2 receptor. Shortening (23) or lengthening (25) the distance betweenthe phenyl ring and the proline core resulted in weaker potency.Compound 24 (NPFF1 K_(e)=850 nM) emerged as a potent selective NPFF1antagonist.

Among electron-donating substituents at the para position of the phenylring (26-31), 4-methoxy (26, NPFF1 K_(e)=670 nM) was the most potentNPFF1 antagonist. 3,4-Dimethoxy (27), and 4-dimethylamino (28) wereslightly less potent. Bulky groups such as 4-acetamido (29),4-(methylamino)carbonyl (30), and t-butyl (31) were not well tolerated,in agreement with the previous observation that there is limited spaceat the binding pocket. Turning to electron-withdrawing substituents,4-nitro (33, NPFF1 K_(e)=250 nM) demonstrated the best NPFF1 antagonistpotency among all proline analogs. The results indicate that strongelectron-withdrawing groups are favored for good NPFF1 antagonistactivity as 3,4-difluoro (34, NPFF1 K_(e)=610 nM) was more potent than4-chloro (32, NPFF1 K_(e)=990 nM), 4-fluoro (35, NPFF1 K_(e)=1,130 nM)and 4-trifluoromethyl (36, NPFF1 K_(e)=1,670 nM). 4-Pyridinyl (37) whichhas been used as an isosteric replacement of 4-nitrophenyl, onlydisplayed moderate NPFF1 activity (K_(e)=1,820 nM).

Finally, the NPFF1 antagonist activity of the two bulkyelectron-withdrawing groups, acetyl (38) and 4-methylsulfonyl (39) wassignificantly dampened compared to the 4-nitro analog. These data implythat small, strong electron-withdrawing substituents were preferredwhereas bulky groups proved to be deleterious for NPFF1 antagonistactivity. Similar to the aliphatic series, these phenethyl analogs werenot potent at the NPFF2 receptor except 4-methoxy (26), diethylamino(28), 4-chloro (32), 4-nitro (33), and 3,4-difluoro (34).

Next, the effect of disubstituted amides at this region was alsoinvestigated. Diethylamino and dipropylamino analogs (40, NPFF1K_(e)=2,900 nM and 41, NPFF1 K_(e)=880 nM) were more potent at the NPFF1receptor compared to their monosubstituted amide counterparts (11, NPFF1K_(e)>10,000 nM and 12, NPFF1 K_(e)=9,080 nM). Compound 42 (NPFF1K_(e)=1,330 nM) with a rigid spacer between the phenyl ring and theamide was less active at the NPFF1 receptor compared to 24 (NPFF1K_(e)=850 nM) with a flexible ethylene linker.

TABLE 2 SAR at the carboxamide region of the 4-(4-methylthio)benzylaminoseries.

Compound R₁ NPFF1 K_(e) (nM)^(a) NPFF2 K_(e) (nM)^(a) 43 Et 1,080 ± 1205,600 ± 590^(b) 44 n-Pentyl  360 ± 50  970 ± 30^(c) 45

 530 ± 80 3,700 ± 940 46

  870 ± 100 2,300 ± 280^(c) 47

370 ± 70^(c) 1,350 ± 200 48

 470 ± 60   880 ± 160^(c) ^(a)Values are the mean ± SEM of at leastthree independent experiments in duplicate. ^(b)Values are the mean ±SME of two independent experiments in duplicate. ^(c)Pre-incubation ofantagonist and test compound was 1 hr.

Since the initial compound 1 has a 4-methylthio at the 4-benzyl group onthe proline scaffold, after exploring various substituents at thecarboxamide region with a 4-(4-methoxybenzyl) substitution of theproline scaffold, several of the more potent analogs from Table 1 wereselected and the effects examined of the 4-methylthio substitution. Asshown in Table 2, the resynthesized 43 (NPFF1 K_(e)=1,080 nM, NPFF2K_(e) 5,600 nM) had comparable potency at both receptors to 1 (NPFF1K_(e)=1,620 nM, NPFF2 K_(e)=7,250 nM) from the screening library. Two(methylthio)benzyl analogs with alkyl groups at the carboxamide weremore potent NPFF1 antagonists than their methoxy counterparts (NPFF1: 43K_(e)=1,080 nM vs. 11 K_(e)>10,000 nM; 44 K_(e)=360 nM vs. 16 K_(e)=720nM). Similarly, phenethyl (45) and 3,4-difluorophenethyl (48) analogsdemonstrated slightly better NPFF1 activities in the thioether seriesthan their methoxy counterparts (45 K_(e)=530 nM vs. 24 K_(e)=850 nM; 48K_(e)=470 nM vs. 34 K_(e)=610 nM). On the other hand, the 4-methoxy (46)and 4-nitro (47) analogs were slightly less potent than their methoxyequivalents (46 K_(e)=870 nM vs. 26 K_(e)=670 nM, 47 K_(e)=370 nM vs. 33K_(e)=250 nM). At the NPFF2 receptor, this series appeared to be moreactive and thus, less selective for NPFF1 receptor, compared to the4-(4-methoxybenzyl)amino analogs except for 46 and 47.

Collectively, these results highlight the importance of the substituentsize and a preference for lipophilicity and some flexibility at thisbinding pocket. The most potent NPFF1 ligand is 4-nitro (33) with aNPFF1 K_(e)=250 nM. Several ligands with moderate activity against NPFF1with no/weak NPFF2 activity were also identified. Throughout the courseof these studies, most of the compounds we tested displayed competitiveantagonism in the curve-shift assays; however, some compounds (23, 32,33, 41, 42, 46) showed evidence of insurmountable antagonism by shiftingthe curve to the right and also depressing the maximal NPFF signal.While allosteric modulators commonly produce such a response, this typeof antagonism can also be observed with competitive orthostericantagonists with slow dissociation rates. Such antagonists have beenworked with previously and showed that by performing the curve-shiftassays with longer antagonist-receptor incubation periods, the systemreaches equilibrium, and hence the compounds produce a typicalcompetitive antagonist profile. Indeed, when the longer incubations wereapplied to the NPFF assays, the compounds displayed the typicalcompetitive antagonist activity profile.

In another embodiment, the NPFF modulators may be represented by FormulaIII:

wherein R₃ is selected from C₃-C₉ alkyl, aryl, heteroaryl, heterocycle,heteroarylalkyl, heterocyclealkyl, and arylalkyl; and R₄ is selectedfrom H and C₁-C₂ alkyl.

In certain embodiments of Formula III, R₃ is substituted benzyl orsubstituted phenethyl with a diversity of substituents. By way ofexample, the substituents may be halogen, methoxy, methyl, cyano, N—CH₂,among others.

In certain embodiments of Formula III, R₃ is benzyl or substitutedbenzyl, phenethyl or substituted phenethyl, and R₄ is H. In someembodiments where R₃ is substituted benzyl, the benzyl is substituted bymethoxy.

In certain other embodiments of Formula III, R₃ is benzyl or substitutedbenzyl and R₄ is methyl.

In other embodiments of Formula III, R₃ is C₃-C₆ alkyl.

Table 3 lists the SAR, determined as discussed above, of analogs ofCompound 1 substituted at region 2, including embodiments correspondingto Formula III.

TABLE 3

Com- NPFF1 NPFF2 pound R₃ R₄ K_(e) (nM) K_(e) (nM) 52 n-Pr H 5,160 ±770   >10,000 53 n-Bu H 6,120 ± 2,200 >10,000 54 n-Pentyl H 2,480 ±280   >10,000 55 n-Hexyl H 2,320 ± 320   4,470 ± 2,110 56

H 1,230 ± 240   1,400 ± 350   57

Me 1,930 ± 180   1,500 ± 810   58

H 1,150 ± 260   1,130 ± 240   33

H 250 ± 60^(b) 690 ± 140^(b) 59

H 1,390 ± 290   2,820 ± 1,420 60

H 1,920 ± 790   3,150 ± 560   61

H 4,790 ± 1,390 6,370 ± 1,700 62

H 2,060 ± 180   6,380 ± 4,670 63

H 680 ± 180 1,860 ± 620   64

H 960 ± 690 >10,000 ^(a)Values are the mean ± SEM of at least threeindependent experiments performed in duplicate. ^(b)Pre-incubation ofantagonist and test compound was 1 hour.

To explore the SARs at the region 2, 4-nitrophenethylamino was selectedas an optimal substituent at R₁ and various substituents were introducedat the amine center at the 4-position of the proline scaffold (Table 3).A similar trend of the substituent size in the aliphatic series asdiscussed above was observed (52-55). As the length of the side chainsincreased from n-propyl to n-hexyl, the NPFF1 activity became morepotent against both receptors. The activity appeared to plateau as theside chain reached 5 to 6 carbons. Phenylmethyl (56) and phenethyl (58)had similar activities against both receptors. N-methylation of theamino group resulting in the tertiary amine analog (57) slightlydampened the activities at both NPFF subtypes. This trend is believed toimply that this binding pocket also has a limited space similar to thatof the region 1.

The effects of substituents at different positions on the benzyl rings(33, 59-64) were further probed. Switching the 4-OMe to 3-position oraddition of another OMe at the 3-position on the phenyl decreasedactivities on both receptor subtypes. The electronic effect did notappear to be a determining factor for the ring substitution. Replacingthe 4-OMe by another electron-donating group 4-OH caused a loss ofactivity. The electron-withdrawing trifluoromethyl group was not activewhile the other two halogen analogs had good activity against NPFF1. Itis noteworthy that compound 64 has more than 10 fold selectivity forNPFF1 receptor.

In another embodiment, the NPFF modulators may be represented by FormulaIV:

wherein R₅ is selected from C₃-C₉ alkyl, heteroarylalkyl, heteroaryl,heterocyclealkyl, heterocycle, cycloalkylalkyl, and arylalkyl; and X isS, SO, SO₂, O, NH or CH₂.

In certain embodiments of Formula IV, R₅ is selected from C₃-C₉ alkyl,heteroarylalkyl, heterocyclealkyl, cycloalkylalkyl, and arylalkyl and Xis O.

In certain embodiments of Formula IV, R₅ is selected from C₃-C₉ alkyl,heteroarylalkyl, heterocyclealkyl, cycloalkylalkyl, and arylalkyl and Xis S.

In certain embodiments of Formula IV, R₅ is selected from C₃-C₉ alkyl,heteroarylalkyl, heterocyclealkyl, cycloalkylalkyl, and arylalkyl and Xis NH.

In some embodiments of Formula I, R₅ is benzyl or substituted benzyl andX is O. In such embodiments where R₅ is substituted benzyl, thesubstituents may be halogen or methoxy. In further embodiments where R₅is substituted benzyl, the benzyl is monosubstituted and thesubstituents may be halogen or methoxy at the 2- or 3-position.

Table 4 lists the SAR, determined as discussed above, of analogs ofCompound 1, substituted at region 3 of compound 1, including embodimentscorresponding to Formula IV.

TABLE 4 NPFF1 NPFF2 Compound R₅ K_(e) (nM)^(a) K_(e) (nM)^(a) 74 n-hexyl1,450 ± 150 7,100 ± 760   75

 650 ± 70 3,640 ± 830   76

1,270 ± 140 >10,000 24

 850 ± 140 9,390 ± 4,670 77

 580 ± 50 4,100 ± 1,490 78

2,150 ± 430 4,850 ± 190   79

1,230 ± 240 5,110 ± 900   80

2,460 ± 390 >10,000 81

9,020 ± 900 5,360 ± 720   82

 490 ± 70 3,940 ± 550   83

1,320 ± 220 >10,000 84

>10,000 >10,000 85

3,510 ± 860 >10,000 86

3,830 ± 260 >10,000 87

1,720 ± 240 7,220 ± 2,360 88

1,860 ± 430 >10,000 ^(a)Values are the mean ± SEM of at least threeindependent experiments in duplicate.

In the synthesis, the nitro group is easily reduced under the conditionsemployed to remove protective groups of the proline nitrogen and theamino group at the 4-position. In order to simplify the synthetic effortto prepare a series of analogs at the region 3, analogs with a phenethylgroup in region 1 were explored, instead of 4-nitrophenylethyl, and the4-methoxybenzyl group was used in region 2.

As illustrated in Table 4, R₅ could be an acyclic (i.e. n-hexyl, 74),cyclic alkyl (i.e. cyclohexylmethyl, 75), or benzyl substituent (24,76-88). The position of the substituents has a strong influence in theactivities. Among the substituted benzyl analogs, substitutions at the2- and 3-positions were preferred to the 4-position. Within the threechloro substituted isomers, substitution at the 3-position is the mostpotent analog (77, K_(e)=580 nM) with a moderate subtype selectivity(>10 fold) over the NPFF2 receptor. On the other hand, the methoxy (79)and methyl (82) groups seems to prefer the 2-position. Similarly, othersubstituents such as 4-hydroxy (84), 4-dimethylamino (85), and 4-cyano(86) groups weakened the NPFF activities. A bulky substituent such as2-naphthylmethyl (87) or 3,4-methylenedioxybenzyl (88) was also notfavored.

Several compounds were further characterized in radioligand binding andcAMP assays (Table 5). In general, the data obtained from all threeassays compare well with each other. Compounds that were potentantagonists in the calcium mobilization assays were also potentantagonists in the secondary cAMP assay that measure native G-proteincoupling. Further, the binding assays showed that all compounds bind tothe NPFF receptors and that potent compounds (e.g., compounds 33 and 34)in the NPFF1 functional assays have potent binding affinities.

TABLE 5 Results from the cAMP, radioligand binding and calciummobilization assays of representative compounds. Calcium mobilizationRadioligand assay cAMP assay^(b) assay^(b) K_(i) K_(i) K_(e) K_(e) K_(e)Compound NPFF1 NPFF2 NPFF1 NPFF2 NPFFI K_(e) NPFF2 # R₁ (nM) (nM) (nM)(nM)^(a) (nM (nM) 16 n-Pentyl 890 ± 50 1,440 ± 130 340 ± 80  2,150 ± 360720 ± 10  3,090 ± 580 24

710 ± 20 1,230 ± 270 770 ± 120 3,790 ± 450 850 ± 140 >10,000^(b) 26

920 ± 30 1,460 ± 50  570 ± 150^(c) 2,560 ± 200 670 ± 60  1,750 ± 110^(c)33

610 ± 30 1,670 ± 60  490 ± 90   1170 ± 360 250 ± 60^(c)   690 ± 140^(c)34

560 ± 40 1,490 ± 270 570 ± 170 2,160 ± 300 610 ± 130 3,490 ± 1,370^(b,c)63

d d 1,552 1,757 510 ± 180 1,860 ± 620 64

d d 1,445 960 960 ± 690 >10,000 ^(a)Values are the mean ± SEM of atleast two independent experiments in duplicate. ^(b)Values are the mean± SEM of at least three independent experiments in duplicate.^(c)Pre-incubation of antagonist and receptor was 1 hour. ^(d)Notdetermined

One of major challenges for CNS drugs is their ability to cross theblood-brain barrier (BBB) and reach the CNS. For a majority of drugs,the blood brain barrier permeability is affected by two factors, theability to permeate through the BBB passively and the avoidance of beingeffluxed out by the transport proteins such as the P-glycoprotein. Thus,representative compound 33 was evaluated, which has good antagonistactivities in the bidirectional transport assay using the MDCK-MDR1cells which are stably transfected with human MDR1 cDNA so that theyexpress a higher level of the P-glycoprotein (Pgp) than the wild type.Compound 33 traversed the cell barrier from the apical (A) tobasolateral (B) at a rate of 2.7××10⁻⁶ cm/s, and the reverse direction Bto A at a rate of 3.6×10⁻⁶ cm/s, demonstrating a moderate BBBpermeability (within the range of 3−6×10⁻⁶ cm/s).³³ The efflux ratio(P_(B→A)/P_(A→B)) is 1.3, indicating that the compound is not a Pgpsubstrate. (Di et al., In Drug-Like Properties: Concepts, StructureDesign and Methods from ADME to Toxicity Optimization, Academic Press,pp. 141-159 (2016)). In addition, the compound also has a solubility45.9±7.7 μM (Mean±% CV) which falls in the range of 10-60 μg/ml forcompounds with moderate to good solubility according to Di et al., InDrug-Like Properties: Concepts, Structure Design and Methods from ADMEto Toxicity Optimization, Academic Press, pp. 56-85 (2016).

The activity shown indicates the proline-based neuropeptide FF receptormodulators are useful as NPFF antagonists.

Terms are used within their accepted meanings. The following definitionsare meant to clarify, but not limit, the terms defined.

As used herein, the singular forms “a”, “and”, and “the” include pluralreferents unless the context clearly dictates otherwise.

As used herein, the identification of a carbon number range, e.g., inC₁-C₁₂ alkyl, is intended to include each of the component carbon numbermoieties within such range, so that each intervening carbon number andany other stated or intervening carbon number value in that statedrange, is encompassed, it being further understood that sub-ranges ofcarbon number within specified carbon number ranges may independently beincluded in smaller carbon number ranges, within the scope of theinvention, and that ranges of carbon numbers specifically excluding acarbon number or numbers are included in the invention, and sub-rangesexcluding either or both of carbon number limits of specified ranges arealso included in the disclosure.

Accordingly, C₃-C₉ alkyl, by way of example, is intended to includepropyl, butyl, pentyl, hexyl, heptyl, octyl, and nonyl, includingstraight chain as well as branched groups of such types, such asisopropyl and tert-butyl. It therefore is to be appreciated thatidentification of a carbon number range, e.g., C₁-C₁₂ or C₁-C₆, asbroadly applicable to a substituent moiety, enables, in specificembodiments of the disclosure, the carbon number range to be furtherrestricted, as a sub-group of moieties having a carbon number rangewithin the broader specification of the substituent moiety. By way ofexample, the carbon number range e.g., C₁-C₁₂ alkyl, may be morerestrictively specified, in particular embodiments of the disclosure, toencompass sub-ranges such as C₁-C₄ alkyl, C₂-C₅ alkyl, C₂-C₄ alkyl,C₃-C₅ alkyl, or any other sub-range within the broad carbon numberrange. Thus, for example, the range C₁-C₆ would be inclusive of and canbe further limited by specification of sub-ranges such as C₁-C₃, C₁-C₄,C₂-C₆, C₄-C₆, etc. within the scope of the broader range.

The term “lower alkyl” includes any of C₁, C₂, or C₃ alkyl.

When the term “alkyl” used as a suffix in conjunction with a secondgroup, as in “arylalkyl”, “aminoalkyl,” “cycloalkylalkyl”, or“heterocyclealkyl” the second group is then connected to the rest of themolecule via an alkyl radical. By way of example, where “arylalkyl”,“aminoalkyl,” “cycloalkylalkyl”, or “heterocyclealkyl” etc., is used,the alkyl radical may be any of C₁, C₂, C₃ or C₄ alkyl.

“Cycloalkyl” refers to an optionally substituted non-aromatic cyclichydrocarbon ring. Unless otherwise indicated, cycloalkyl is composed ofthree to eight carbon atoms. Exemplary “cycloalkyl” groups include, butare not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,and cycloheptyl. The cycloalkyl group may be substituted orunsubstituted, for, example by a halogen, or a C₁-C₃-alkyl.

“Heterocycle” refers to saturated cyclic radicals containing one or moreheteroatoms (e.g., 0, N, S) as part of the ring structure and having twoto seven carbon atoms in the ring. In embodiments, a heterocycle may befused to an aryl group such as phenyl. The heterocycle may besubstituted or unsubstituted, for example, by a halogen, amino, cyano,nitro, carbonyl, amido, acetyl, carboxymethyl, C₁-C₃-alkyl or C₁-C₃alkoxy.

“Heteroaryl” refers to unsaturated aromatic cyclic radicals containingone or more heteroatoms (e.g., O, N, S) as part of the ring structureand having two to seven carbon atoms in the ring. Heteroaryl groups mayinclude furanyl, thienyl, pyridyl, pyrrolyl, pyrrolo, pyrimidyl,pyrazinyl, imidazolyl and the like. The heteroaryl group may besubstituted or unsubstituted, for, example, by a halogen, amino, cyano,nitro, carbonyl, amido, acetyl, carboxymethyl, C₁-C₃-alkyl or C₁-C₃alkoxy.

In addition, the term “heteroaryl” is used to include fused bicyclicring systems that contain one or more heteroatoms. Examples of suchheteroaryl groups include benzodioxole, benzisoxazolyl, benzofuranyl.

“Aryl” as used herein includes hydrocarbons derived from benzene or abenzene derivative that are unsaturated aromatic carbocyclic groups offrom 6 to 10 carbon atoms. The aryls may have a single or multiplerings. Examples include phenyl, benzyl, or naphthyl. The aryl group maybe substituted or unsubstituted, for example, by a halogen, amino,cyano, nitro, carbonyl, amido, acetyl, carboxymethyl, C₁-C₃-alkyl orC₁-C₃ alkoxy.

“Arylalkyl” includes, by way of example, benzyl and phenethyl.

“Alkylcycloalkyl” includes, by way of example, a methylcylohexyl group.

The compounds of the disclosure may be further specified in specificembodiments by provisos or limitations excluding specific substituents,groups, moieties or structures, in relation to various specificationsand exemplifications thereof set forth herein. Thus, the disclosurecontemplates restrictively defined compositions, e.g., a compositionwherein R is C₁-C₁₂ alkyl, with the proviso that R≠ C_(i) alkyl when R₁is a specified molecular component, and i is a specific carbon number.The substituents maybe selected and combined with each other in anymanner resulting in a compound according to Formulas I, II, IIA, III orIV.

Certain of the compounds disclosed herein may exist as stereoisomersincluding optical isomers. Scope of the disclosure includes allstereoisomers in both the racemic mixtures of such stereoisomers as wellas the individual enantiomers which may be separated according tomethods that are well known to those of ordinary skill in the art. Whenchiral centers are present, the stereochemistry of the structuresincludes both R and S configuration, unless otherwise indicated.

The disclosure, as variously set out herein in respect of variousdescribed features, aspects and embodiments, may in particularimplementations be constituted as comprising, consisting, or consistingessentially of, some or all of such features, aspects and embodiments,as well as elements and components thereof being aggregated toconstitute various further implementations of the disclosure. Thedisclosure contemplates such features, aspects and embodiments invarious permutations and combinations, as being within the scope of thedisclosure. The disclosure may therefore be specified as comprising,consisting or consisting essentially of, any of such combinations andpermutations of these specific features, aspects and embodiments, or aselected one or ones thereof.

The term “effective amount” means that amount of a drug orpharmaceutical agent that will elicit the biological or medical responseof a tissue, system, animal, or human that is being sought, forinstance, by a researcher or clinician. The term “therapeuticallyeffective amount” means any amount which, as compared to a correspondingsubject who has not received such amount, results in improved treatment,healing, prevention, or amelioration of a disease, disorder, or sideeffect, or a decrease in the rate of advancement of a disease ordisorder. The term also includes within its scope amounts effective toenhance normal physiological function.

The NPFF receptor modulators may be useful in pain management to reduceopioid tolerance and hyperalgesia, addiction disorders,anti-inflammation, feeding, blood pressure, insulin release, attenuationof fever, reduction of anxiety, attenuation of limbic seizure activity,attenuation of opioid-induced hypothermia, and cardiovascularmodulation, by way of example.

In one aspect, the NPFF receptor modulators may be used in combinationwith other drugs or agents, or in conjunction with a variety ofpsychotherapies useful in the treatment of the type of conditions anddisorders modulated by NPFF receptors. Drugs or agents which may be usedwith the NPFF receptor modulators and compositions containing same mayinclude typical and/or atypical antipsychotics such as haloperidol andaripiperazole or monoamine reuptake inhibitors such as fluoxetine andsertraline.

In view of the opioid-modulating properties of the NPFF system, othercombination therapy applications of the NPFF receptor modulators andcompositions of the present disclosure include their contemporaneousadministration with opioids, e.g., fentanyl, morphine, oxycodone,hydrocodone, buprenorphine, etc., to combat hyperalgesia and toleranceeffects of the opioids.

In another aspect of the disclosure, a method for treating a subjecthaving or susceptible to a condition or disorder where modulation ofneuropeptide FF receptor activity is of therapeutic benefit is provided,the method comprising administering to said subject having orsusceptible to said condition or disorder an effective amount of aneuropeptide FF modulator according to Formula I:

wherein R₂ is selected from —N—(C₂-C₅alkyl)₂, and NH—R₁, wherein R₁ isselected from C₂-C₉ alkyl, heterocyclealkyl, cycloalkylalkyl,aminoalkyl, and arylalkyl; R₃ is selected from C₃-C₉ alkyl, aryl,heteroaryl, heterocycle, heteroarylalkyl, heterocyclealkyl, andarylalkyl; R₄ is selected from H and C₁-C₂ alkyl; and R₅ is selectedfrom C₃-C₉ alkyl, heteroarylalkyl, heteroaryl, heterocyclealkyl,heterocycle, cycloalkylalkyl, and arylalkyl; or a pharmaceutical salt,amide, ester or prodrug thereof.

In embodiments of Formula I, wherein R₁ is selected fromheterocyclealkyl, cycloalkylalkyl, aminoalkyl, and arylalkyl; R₃ isselected from heteroarylalkyl, heterocyclealkyl, and arylalkyl; and/orR₅ is selected from heteroarylalkyl, heterocyclealkyl, cycloalkylalkyl,and arylalkyl, the alkyl group is C₁, C₂ or C₃. Suitable examples ofsuch groups are shown in the Tables above.

In certain embodiments of Formula I, R₂ is NH—R₁. In further embodimentsof Formula I, when R₂ is NH—R₁, R₁ is C₃-C₆ alkyl.

In some other embodiments of Formula I, when R₂ is NH—R₁, R₁ is benzylor phenethyl, substituted or unsubstituted. In certain embodiments whereR₁ is substituted phenethyl, the phenethyl is substituted by loweralkoxy such as methoxy, nitro, lower alkyl, halogen, or halogenatedlower alkyl such as CF₃. In certain embodiments, the phenethyl group ismonosubstituted or disubstituted.

Embodiments of R₂ as described herein may be combined with anycombination of embodiments of R₃, R₄ and R₅ described herein.

In certain embodiments of Formula I, R₃ is substituted benzyl orsubstituted phenethyl with a diversity of substituents. By way ofexample, the substituents may be halogen, methoxy, methyl, cyano, N—CH₂,among others.

In certain embodiments of Formula I, R₃ is benzyl or substituted benzyland R₄ is H. In such embodiments where R₃ is substituted benzyl, thebenzyl is mono- or di-substituted with methoxy.

In certain other embodiments of Formula I, R₃ is benzyl or substitutedbenzyl and R₄ is methyl.

In embodiments of Formula I where R₃ is monosubstituted benzyl orphenethyl, the substituent is at the 4-position.

In some embodiments of Formula I, R₅ is benzyl or substituted benzyl. Insuch embodiments where R₅ is substituted benzyl, the substituents may behalogen or methoxy. In further embodiments where R₅ is substitutedbenzyl, the benzyl is monosubstituted and the substituents may beortho-substituted halogen or methoxy.

In one embodiment, the proline-based NPFF modulators may be representedby Formula II:

wherein R₂ is selected from —N—(C₂-C₅alkyl)₂, and NH—R₁, wherein R₁ isselected from C₂-C₉ alkyl, heterocyclealkyl, cycloalkylalkyl,aminoalkyl, and arylalkyl; and X is S, SO, SO₂, O, NH or CH₂.

According to some embodiments of Formula II, R₂ is NH—R₁, wherein R₁ isselected from C₂-C₉ alkyl, heterocyclealkyl, cycloalkylalkyl,aminoalkyl, and arylalkyl; and X is S, SO, SO₂, O, NH or CH₂. Thisembodiment is represented by Formula IIA:

In certain embodiments of Formula IIA, R₁ is C₃-C₆ alkyl; and X isoxygen. In other embodiments of Formula IIA, R₁ is benzyl or phenethyl,substituted or unsubstituted and X is oxygen. In certain embodimentswhere R₁ is substituted phenethyl, the phenethyl is substituted by loweralkoxy, such as methoxy, nitro, lower alkyl, halogen, or halogenatedlower alkyl such as CF₃.

In other embodiments of the method for treating a subject, the NPFFreceptor modulators may be according to any of the embodiments disclosedherein for Formulas I, II or IIA.

In certain embodiments of the method for treating a subject, the NPFFreceptor modulators may be according to any of Formulas III or IV asdescribed herein.

In certain embodiments of the method for treating a subject, themodulation of neuropeptide FF receptor activity is antagonisticactivity.

In a certain embodiment, a method is provided for treating a subjecthaving a condition or disorder where modulation of neuropeptide Sreceptor activity is of therapeutic benefit comprising administering tosaid subject having or susceptible to said condition or disorder aneffective amount of a compound according to one of Formulas I, II, IIA,III or IV demonstrating selective binding and functional antagonistactivity at a neuropeptide FF receptor.

The condition or disorder to be treated may be related to painmanagement, addiction disorders, anti-inflammation, feeding, bloodpressure, insulin release, attenuation of fever, reduction of anxiety,attenuation of limbic seizure activity, attenuation of opioid-inducedhypothermia, cardiovascular modulation, attenuation of hyperalgesiaand/or opioid tolerance or other conditions or disorder modulated by theNPFF receptors.

In embodiments, the NPFF receptor modulator or antagonist administeredis a pharmaceutically acceptable salt, amide, ester or prodrug of anymodulators of the foregoing formulas I, II, IIA, III or IV. In thisaspect, any of the compounds of Formulas I, II, IIA, III or IV may becombined with a pharmaceutically acceptable carrier.

Salts of the compounds of the present disclosure may be made by methodsknown to a person skilled in the art. The acid may be an inorganic acidor an organic acid. Suitable acids include, for example, hydrochloric,hydroiodic, hydrobromic, sulfuric, phosphoric, citric, acetic and formicacids.

A variety of administration techniques may be utilized, among them oral,transdermal or parenteral techniques such as subcutaneous, intravenous,intraperitoneal, intracerebral and intracerebroventricular injections,catheterizations and the like. Such methods of administration arewell-known to those skilled in the art. For a general discussion of drugdelivery systems and administration modalities, see Kirk-OthmerEncyclopedia of Chemical Technology, Fourth Edition, Volume 8, pp.445-475.

Average quantities of the compounds may vary in accordance with thebinding properties of the compound (i.e., affinity, onset and durationof binding) and in particular should be based upon the recommendationsand prescription of a qualified physician.

The therapeutic compositions useful in practicing the therapeuticmethods of this disclosure may include, in admixture, a pharmaceuticallyacceptable excipient (carrier) and one or more of the NPFF receptormodulators, as described herein as an active ingredient.

The NPFF receptor ligands may be administered by a variety of methods.Thus, those products of the invention that are active by the oral routemay be administered in solutions, suspensions, emulsions, tablets,including sublingual and intrabuccal tablets, soft gelatin capsules,including solutions used in soft gelatin capsules, aqueous or oilsuspensions, emulsions, pills, lozenges, troches, tablets, syrups orelixirs and the like. NPFF receptor modulators active on parenteraladministration may be administered by depot injection, implantsincluding Silastic™ and biodegradable implants, skin patches, skincreams, or intramuscular and intravenous injections.

Compositions containing the NPFF receptor modulators may be preparedaccording to any method known to the art for the manufacture ofpharmaceutical compositions and such compositions may contain one ormore agents selected from the group consisting of sweetening agents,flavoring agents, coloring agents and preserving agents. Tabletscontaining the active ingredient in admixture with nontoxicpharmaceutically acceptable excipients which are suitable formanufacture of tablets are acceptable. These excipients may be, forexample, inert diluents, such as calcium carbonate, sodium carbonate,lactose, calcium phosphate or sodium phosphate; granulating anddisintegrating agents, such as maize starch, or alginic acid; bindingagents, such as starch, gelatin or acacia; and lubricating agents, suchas magnesium stearate, stearic acid or talc. Tablets may be uncoated ormay be coated by known techniques to delay disintegration and adsorptionin the gastrointestinal tract and thereby provide a sustained actionover a longer period. For example, a time delay material such asglyceryl monostearate or glyceryl distearate alone or with a wax may beemployed.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, such as peanut oil, liquid paraffin or olive oil.

Aqueous suspensions of the disclosure contain the active materials inadmixture with excipients suitable for the manufacture of aqueoussuspensions. Such excipients include a suspending agent, such as sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylethyl cellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia,and dispersing or wetting agents such as a naturally occurringphosphatide (e.g., lecithin), a condensation product of an alkyleneoxide with a fatty acid (e.g., polyoxyethylene stearate), a condensationproduct of ethylene oxide with a long chain aliphatic alcohol (e.g.,heptadecaethyleneoxycetanol), a condensation product of ethylene oxidewith a partial ester derived from a fatty acid and a hexitol (e.g.,polyoxyethylene sorbitol mono-oleate), or a condensation product ofethylene oxide with a partial ester derived from a fatty acid and ahexitol anhydride (e.g., polyoxyethylenesorbitan mono-oleate). Theaqueous suspension may also contain one or more preservatives such asethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one ormore flavoring agents and one or more sweetening agents, such assucrose, aspartame or saccharin. Ophthalmic formulations, as is known inthe art, will be adjusted for osmotic pressure.

Oil suspensions may be formulated by suspending the active ingredient ina vegetable oil, such as arachis oil, olive oil, sesame oil or coconutoil, or in a mineral oil such as liquid paraffin. The oil suspensionsmay contain a thickening agent, such as beeswax, hard paraffin or cetylalcohol. Sweetening agents may be added to provide a palatable oralpreparation. These compositions may be preserved by the addition of anantioxidant such as ascorbic acid.

Dispersible powders and granules of the disclosure suitable forpreparation of an aqueous suspension by the addition of water may beformulated from the active ingredients in admixture with a dispersing,suspending and/or wetting agent, and one or more preservatives. Suitabledispersing or wetting agents and suspending agents are exemplified bythose disclosed above. Additional excipients, for example sweetening,flavoring and coloring agents, may also be present.

The pharmaceutical composition may also be in the form of oil-in-wateremulsions. The oily phase may be a vegetable oil, such as olive oil orarachis oil, a mineral oil, such as liquid paraffin, or a mixture ofthese. Suitable emulsifying agents include naturally occurring gums,such as gum acacia and gum tragacanth, naturally occurring phosphatides,such as soybean lecithin, esters or partial esters derived from fattyacids and hexitolamhydrides, such as sorbitan mono-oleate, andcondensation products of these partial esters with ethylene oxide, suchas polyoxyethylenesorbitan mono-oleate. The emulsion may also containsweetening and flavoring agents.

Syrups and elixirs may be formulated with sweetening agents, such asglycerol, sorbitol or sucrose. Such formulations may also contain ademulcent, a preservative, a flavoring or a coloring agent.

The pharmaceutical compositions may be in the form of a sterileinjectable preparation, such as a sterile injectable aqueous oroleaginous suspension. This suspension may be formulated according tothe known art using those suitable dispersing or wetting agents andsuspending agents which have been mentioned above. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a nontoxic parenterally acceptable diluent or solvent,such as a solution of 1,3-butanediol. Among the acceptable vehicles andsolvents that may be employed are water and Ringer's solution, anisotonic sodium chloride solution. In addition, sterile fixed oils mayconventionally be employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed including synthetic mono- ordiglycerides.

In addition, fatty acids such as oleic acid may likewise be used in thepreparation of injectables. Sterilization may be performed byconventional methods known to those of ordinary skill in the art suchas, for example, by aseptic filtration, or irradiation.

Aqueous formulations (i.e., oil-in-water emulsions, syrups, elixirs andinjectable preparations) may be formulated to achieve the pH of optimumstability. The determination of the optimum pH may be performed byconventional methods known to those of ordinary skill in the art.Suitable buffers may also be used to maintain the pH of the formulation.

The NPFF receptor modulators may also be administered in the form ofsuppositories for rectal administration of the drug. These compositionscan be prepared by mixing the drug with a suitable nonirritatingexcipient which is solid at ordinary temperatures but liquid at rectaltemperatures and will therefore melt in the rectum to release the drug.Non-limiting examples of such materials are cocoa butter andpolyethylene glycols.

They may also be administered by intranasal, intraocular, intravaginal,and intrarectal routes including suppositories, insufflation, powdersand aerosol formulations.

Products as disclosed herein which are preferably administered by thetopical route may be administered as applicator sticks, solutions,suspensions, emulsions, gels, creams, ointments, pastes, jellies,paints, powders, and aerosols.

The compositions may, if desired, the person or dispenser device whichmay contain one or more unit dosage forms containing an activeingredient. Pharmaceutical compositions comprising an NPFF modulator asdescribed herein and formulated in a compatible pharmaceutical carriermay also be prepared, placed in an appropriate container, and labeledfor treatment of an indicated condition.

The advantages and features of the disclosure are further illustratedwith reference to the following examples, which are not to be construedas in any way limiting the scope of the disclosure but rather asillustrative of embodiments of the disclosure.

EXAMPLES

Chemistry. Compounds 10-48 were synthesized following proceduresdepicted in Scheme 1. trans-4-Hydroxy-L-proline methyl ester (2)underwent reductive amination by sodium triacetoxyborohydride in1,2-dichloroethane to give the intermediate 3 which was then convertedto a tosylated derivative 4 in the presence of pyridine indichloromethane. S_(N)2 substitution with sodium azide provided azide 5which subsequently underwent Staudinger reduction withtriphenylphosphine to give amine 6. A second reductive amination yielded7a and 7b, respectively. This three-step conversion from the tosylate 4to amines 7a-b gave higher yields than the direct displacement with4-methoxybenzylamine or 4-(methylthio)benzylamine. The free amino groupwas then protected with a Boc group and the resulted intermediates 8a-bwere subsequently hydrolyzed under basic conditions to give acids 9a-b.HBTU-assisted amide coupling between 9a-b and corresponding aminesfollowed by cleavage of the Boc protective group provided the finalproducts (10-48). All target compounds were characterized by ¹H and ¹³CNMR, MS and HPLC.

Reagents and conditions for Scheme 1 were as follows: (a)2-chlorobenzaldehyde, Na(OAc)₃BH, 1,2-DCE (1,2-dichloroethane), rt, 24 h(b) TsCl, pyridine, DCM (dichloromethane), rt, 24 h (c) NaN₃, DMF, 70°C., 16 h (d) PPh₃, THF, H₂O, reflux, 16 h (e) 4-MeOPhCHO or 4-MeSPhCHO,Na(OAc)₃BH, 1,2-DCE, rt, 24 h (f) Boc₂O, aq. Na₂CO₃, 1,4-dioxane, rt, 16h (g) aq. LiOH, MeOH, rt, 16 h (h) corresponding amine, HBTU, DIEA, DMF,rt, 24 h (i) TFA, DCM, rt, 1 h.

Compounds 52-654 were obtained via a slightly different route (Scheme2). The ester 3 was hydrolyzed to give acid 49. HBTU-assisted amidecoupling of 49 with 4-nitrophenylethylamine yielded the intermediate 50.50 was converted to the tosylated derivative 51 before undergoing S_(N)2displacement with corresponding amines to give the final products 52-64.The low yields of this S_(N)2 substitution were improved upon additionof more amine equivalents. However, the excess unreacted aminescomplicated the purification of the final product.

Scheme 3 illustrates the synthesis of compounds 74-88.Trans-4-hydroxy-L-proline methyl ester (5) was N-Boc protected as theintermediate 65 and converted to 4-tosyl derivative 66 which underwentS_(N)2 displacement with sodium azide (67) and Staudinger reduction toprovide the intermediate 68. Reductive amination with4-methoxybenzaldehyde by sodium triacetoxyboron hydride give thesecondary amine 69 which was subsequently protected with an N-Trocprotective group as the intermediate 70. Hydrolysis of the methyl ester(71) followed by HBTU-assisted amide coupling with phenethylamine led tointermediate 72. Removal of the N-Boc group by TFA yielded theintermediate 73, which was used to prepare a series of analogs at theregion 3. These analogs (74-88) were attained by reductive amination of73 with corresponding aldehydes and removal of the N-Troc group by Zn inthe presence of acetic acid in methanol under reflux.

Experimental Section

Chemistry. All solvents and chemicals were reagent grade. Unlessotherwise mentioned, all reagents and solvents were purchased fromcommercial vendors and used as received. Flash column chromatography wascarried out on a Teledyne ISCO CombiFlash Rf system using prepackedcolumns. Solvents used include hexane, ethyl acetate (EtOAc),dichloromethane, methanol, and chloroform/methanol/ammonium hydroxide(80:18:2) (CMA-80). Purity and characterization of compounds wereestablished by a combination of HPLC, TLC, mass spectrometry, and NMRanalyses. ¹H and ¹³C NMR spectra were recorded on a Bruker AvanceDPX-300 (300 MHz) spectrometer and were determined in CDCl₃, DMSO-d6, orCD₃OD with tetramethylsilane (TMS) (0.00 ppm) or solvent peaks as theinternal reference. Chemical shifts are reported in ppm relative to thereference signal, and coupling constant (J) values are reported in hertz(Hz). Thin layer chromatography (TLC) was performed on EMD precoatedsilica gel 60 F254 plates, and spots were visualized with UV light oriodine staining. Low resolution mass spectra were obtained using aWaters Alliance HT/Micromass ZQ system (ESI). All test compounds weregreater than 95% pure as determined by HPLC on an Agilent 1100 systemusing an Agilent Zorbax SB-Phenyl, 2.1 mm×150 mm, 5 m, column withgradient elution using the mobile phases (A) H₂O containing 0.1% CF₃COOHand (B) MeCN, with a flow rate of 1.0 mL/min.

Methyl(2S,4R)-1-[(2-chlorophenyl)methyl]-4-hydroxypyrrolidine-2-carboxylate(3). To a solution of methyl trans-4-hydroxy-L-proline (16.5 mmol, 3.00g) and 2-chlorobenzaldehyde (21.5 mmol, 2.1 ml) in 1,2-dichloroethane(55 ml) was added acetic acid (0.9 ml) and sodium triacetoxyborohydride(24.8 mmol, 5.26 g). The reaction was stirred at room temperature for 24h. After quenching with saturated solution of sodium bicarbonate, thereaction mixture was extracted three times with dichloromethane. Thecombined organic layers were washed sequentially with water and brine,dried over anhydrous magnesium sulfate, filtered, and concentrated invacuo. The residue was purified by column chromatography to give thedesired product as yellow liquid (4.00 g, 90%). ¹H NMR (300 MHz, CDCl₃)δ 7.44 (dd, J=1.98, 7.44 Hz, 1H), 7.34 (m, 1H), 7.20 (m, 2H), 4.43 (m,1H), 3.92 (m, 2H), 3.63-3.74 (m, 4H), 3.35 (dd, J=5.56, 10.08 Hz, 1H),2.52 (dd, J=3.58, 10.17 Hz, 1H), 2.24 (m, 1H), 2.12 (m, 1H). MS (ESI)[M]⁺270.2.

Methyl(2S,4R)-1-[(2-chlorophenyl)methyl]-4-[(4-methylbenzenesulfonyl)oxy]pyrrolidine-2-carboxylate(4). To a solution of 13821-125 (14.8 mmol, 4.00 g) in pyridine (11.4ml) and anhydrous dichloromethane (11.4 ml) at 0° C. was added dropwisetosyl chloride (17.8 mmol, 3.39 g). The reaction was refluxed for 24 h.After removal of the solvent in vacuo, the residue was dissolved indichloromethane and washed with saturated copper sulfate, water, andbrine. The combined organic layers were dried over anhydrous magnesiumsulfate, filtered, and concentrated in vacuo. The residue was purifiedby column chromatography (silica gel, ethyl acetate/hexanes) to providethe desired product as colorless liquid (3.77 g, 60%). ¹H NMR (300 MHz,CDCl₃) δ 7.74-7.79 (m, 2H), 7.37-7.46 (m, 1H), 7.28-7.35 (m, 3H),7.17-7.23 (m, 2H), 5.01 (d, J=5.46 Hz, 1H), 3.74-4.04 (m, 2H), 3.69 (s,1H), 3.66 (s, 3H), 3.29 (dd, J=6.03, 11.11 Hz, 1H), 2.67-2.73 (m, 1H),2.44 (s, 3H), 2.28 (dd, J=5.46, 7.54 Hz, 2H). MS (ESI) [M]⁺424.2.

Methyl(2S,4S)-4-azido-1-[(2-chlorophenyl)methyl]pyrrolidine-2-carboxylate (5).To a solution of 4 (6.87 mmol, 2.91 g) in DMF (40 ml) was added sodiumazide (13.74 mmol, 0.89 g). After stirring at 65° C. for 16 h, thereaction mixture was diluted with water, and extracted three times withethyl acetate. The combined organic layers were dried over anhydrousmagnesium sulfate, filtered, and concentrated in vacuo. The residue waspurified by column chromatography (SiO₂, hexanes/ethyl acetate) to givethe desired product as yellow liquid (1.47 g, 73%). ¹H NMR (300 MHz,CDCl₃) δ 7.54 (dd, J=1.70, 7.54 Hz, 1H), 7.34 (dd, J=1.51, 7.72 Hz, 1H),7.16-7.29 (m, 2H), 4.02-4.09 (m, 1H), 3.90-3.98 (m, 1H), 3.81-3.87 (m,1H), 3.72 (s, 3H), 3.45 (dd, J=6.03, 9.23 Hz, 1H), 3.13 (dd, J=1.51,10.36 Hz, 1H), 2.71 (dd, J=5.75, 10.27 Hz, 1H), 2.54 (ddd, J=7.72, 9.23,14.13 Hz, 1H), 2.12-2.21 (m, J=0.90, 3.20, 6.10, 14.10 Hz, 1H) MS (ESI)[M]⁺295.1.

Methyl(2S,4S)-4-amino-1-[(2-chlorophenyl)methyl]pyrrolidine-2-carboxylate (6).To a solution of azide 5 (4.3 mmol, 1.27 g) in THF (19 ml) undernitrogen was added PPh₃ (8.6 mmol, 2.26 g) and water (0.2 ml). Thereaction mixture was refluxed with stirring for 6 h. After the solventwas removed, the residue was dissolved in diethyl ether, treated with0.1 N HCl for 5 min, and then extracted twice with diethyl ether. Theaqueous layer was then treated with 1 N NaOH until pH 10, and thenextracted with dichloromethane. The combined dichloromethane fractionswere dried over anhydrous MgSO₄, concentrated in vacuo to afford thedesired product as yellow liquid (1 g, 86%). ¹H NMR (300 MHz, CDCl₃) δ7.50 (dd, J=1.9, 7.54 Hz, 1H), 7.33 (m, 1H), 7.16-7.20 (m, 2H), 3.95 (d,J=14.7 Hz, 1H), 3.81 (d, J=13.9 Hz, 1H), 3.66 (s, 3H), 3.45 (m, 1H),3.39 (dd, J=5.5, 9.6 Hz, 1H), 2.85 (m, 1H), 2.68 (dd, J=5.5, 9.4 Hz,1H), 2.48 (m, 1H), 1.79 (m, 1H), 1.74 (br, 2H). MS (ESI) [M+H]⁺: 269.3.

Methyl(2S,4S)-1-[(2-chlorophenyl)methyl]-4-{[(4-methoxyphenyl)methyl]amino}pyrrolidine-2-carboxylate(7a). To a solution of amine 6 (3.72 mmol, 1 g) in 1,2-dichloroethane(12.4 ml) was added 4-methoxybenzaldehyde (3.72 mmol, 0.45 ml), sodiumtriacetoxyborohydride (5.58 mmol, 1.18 g) and glacial acetic acid (3.72mmol, 0.21 ml). After stirring at room temperature for 16 h, thereaction was quenched with saturated sodium bicarbonate, extracted threetimes with dichloromethane. The combined organic layers were dried overanhydrous magnesium sulfate, filtered, and concentrated in vacuo. Theresidue was purified by column chromatography (SiO₂,dichloromethane/methanol) to give the desired product as colorlessliquid (0.68 g, 47%). ¹H NMR (300 MHz, CDCl₃) δ 7.49 (dd, J=1.79, 7.44Hz, 1H), 7.33 (m, 1H), 7.16-7.25 (m, 4H), 6.84 (m, 2H), 3.95 (d, J=14.30Hz, 1H), 3.78-3.84 (m, 4H), 3.67 (d, J=3.20 Hz, 5H), 3.39 (dd, J=6.03,9.04 Hz, 1H), 3.28 (m, 1H), 3.03 (dd, J=2.54, 9.51 Hz, 1H), 2.61 (dd,J=6.12, 9.51 Hz, 1H), 2.40 (m, 1H), 1.93 (m, 1H). MS (ESI) [M+H]⁺ 389.4.

Methyl(2S,4S)-1-[(2-chlorophenyl)methyl]-4-{[(4-(methylsulfanyl)phenyl)methyl]amino}pyrrolidine-2-carboxylate (7b) was synthesized from 6 and4-(methylthio)benzylaldehyde according to the procedure for thesynthesis of 7a as yellow liquid (450% yield). ¹H NMR (300 MHz, CDCl₃) δ7.42 (br. s., OH), 7.32 (d, J=6.78 Hz, 0H), 7.13-7.24 (m, 6H), 3.90 (d,J=7.91 Hz, 1H), 3.66 (s, 3H), 3.64 (br. s., 1H), 3.22-3.30 (m, 1H),2.87-2.97 (m, 1H), 2.47-2.50 (m, 2H), 2.46 (s, 3H), 2.02-2.12 (m, 1H).MS (ESI) [M+H]⁺ 405.3.

Methyl(2S,4S)-4-{[(tert-butoxy)carbonyl][(4-methoxyphenyl)methyl]amino}-1-[(2-chlorophenyl)methyl]pyrrolidine-2-carboxylate(8a). To a solution of amine 7 (1.75 mmol, 0.68 g) in 1,4-dioxane (10ml) was added 10% w/v Na₂CO₃ solution (2 ml) and Boc₂O (1.92 mmol, 0.42g). After stirring at room temperature after 16 h, 1,4-dioxane wasremoved under reduced pressure. The remaining aqueous solution wasextracted three times with dichloromethane. The combined organic layerswere dried over anhydrous MgSO₄, filtered, and concentrated in vacuo andthe crude was used for the next step without further purification. ¹HNMR (300 MHz, CDCl₃) δ 7.30-7.38 (m, 2H), 7.16-7.22 (m, 2H), 7.06 (d,J=8.67 Hz, 2H), 6.80 (d, J=8.67 Hz, 2H), 4.47-4.63 (m, 1H), 4.26-4.44(m, 2H), 3.83 (d, J=6.97 Hz, 2H), 3.78 (s, 3H), 3.65 (s, 3H), 3.61 (d,J=6.97 Hz, 1H), 3.06 (t, J=8.29 Hz, 1H), 2.67 (dd, J=7.44, 8.95 Hz, 1H),2.13-2.25 (m, 2H), 1.53 (s, 9H). MS (ESI) [M]⁺489.6.

Methyl(2S,4S)-4-{[(tert-butoxy)carbonyl][(4-(methylsulfanyl)phenyl)methyl]amino}-1-[(2-chlorophenyl)methyl]pyrrolidine-2-carboxylate(8b) was synthesized from 7b according to the procedure for thesynthesis of 8a as yellow liquid (quant. yield). ¹H NMR (300 MHz, CDCl₃)δ 7.34 (d, J=6.22 Hz, 1H), 7.26-7.32 (m, 1H), 7.08-7.19 (m, 4H),7.01-7.06 (m, 2H), 4.79-5.03 (m, 1H), 4.58 (s, 2H), 3.91 (d, J=13.75 Hz,1H), 3.64 (s, 3H), 3.58 (d, J=13.94 Hz, 1H), 3.27 (t, J=8.29 Hz, 1H),2.87-2.96 (m, 1H), 2.49-2.60 (m, 2H), 2.45 (s, 3H), 1.87-1.99 (m, 1H),1.34 (s, 9H). MS (ESI) [M]⁺505.6.

(2S,4S)-4-{[(tert-butoxy)carbonyl][(4-methoxyphenyl)methyl]amino}-1-[(2-chlorophenyl)methyl]pyrrolidine-2-carboxylicacid (9a). To a solution of ester 8 (1.75 mmol, 0.86 g) in methanol (16ml) and water (16 ml) was added LiOH (8.75 mmol, 0.21 g). After stirringat room temperature for 3 h, methanol was removed under reducedpressure. The remaining solution was diluted in water and acidified with1N HCl to pH 5. The mixture was then extracted with ethyl acetate. Thecombined organic layers were washed with water and brine, dried overanhydrous MgSO₄, filtered, and concentrated in vacuo to provide thedesired product as white solid (0.83 g, quantitative yield). ¹H NMR (300MHz, CDCl₃) δ 7.35 (d, J=8.10 Hz, 2H), 7.17-7.24 (m, 2H), 7.02 (d,J=8.48 Hz, 2H), 6.77 (d, J=8.48 Hz, 2H), 4.26-4.55 (m, 3H), 4.06-4.17(m, 1H), 3.71-3.82 (m, 4H), 3.40-3.52 (m, 1H), 3.08 (d, J=6.40 Hz, 1H),2.70-2.82 (m, 1H), 2.44-2.58 (m, 1H), 2.07-2.17 (m, 1H), 1.40 (s, 9H).MS (ESI) [M]⁺475.7, [M−H]⁻ 473.8.

(2S,4S)-4-{[(tert-butoxy)carbonyl][(4-(methylsulfanyl)phenyl)methyl]amino}-1-[(2-chlorophenyl)methyl]pyrrolidine-2-carboxylicacid (9b) was synthesized from 8b according to the procedure for thesynthesis of 9a as white solid (quant. yield). ¹H NMR (300 MHz, CDCl₃) δ7.42 (br. s., 1H), 7.34 (d, J=7.72 Hz, 1H), 7.16-7.26 (m, 2H), 7.13 (d,J=8.10 Hz, 2H), 7.02 (d, J=8.10 Hz, 2H), 4.47-4.61 (m, 1H), 4.31-4.46(m, 2H), 4.23 (d, J=10.93 Hz, 1H), 3.77-3.88 (m, 1H), 3.58 (d, J=10.74Hz, 1H), 3.02-3.13 (m, 1H), 2.81 (d, J=10.36 Hz, 1H), 2.52-2.66 (m, 1H),2.44 (s, 3H), 2.06-2.17 (m, 1H), 1.38 (s, 8H). MS (ESI) [M−H]⁻ 489.5.

General procedure A: To a solution of acid 9 (0.2 mmol, 1 eq) in DMF(0.6 ml, 0.3 M) was added HBTU (0.22 mmol, 1.1 eq), corresponding amine(0.22 mmol, 1.1 eq), DIEA (0.66 mmol, 3 eq). After stirring at roomtemperature for 16 h, the reaction mixture was diluted with water,extracted three times with ethyl acetate. The combined organic fractionswere dried over anhydrous magnesium sulfate and concentrated in vacuo.The residue was dissolved in 20% v/v trifluoroacetic acid (1 ml) indichloromethane and stirred at room temperature for 16 h. The reactionmixture was concentrated under reduced pressure and the residue waspurified on column chromatography (SiO₂, methanol/dichloromethane) toafford the desired product.

(2S,4S)-1-[(2-Chlorophenyl)methyl]-4-{[(4-methoxyphenyl)methyl]amino}-N-methylpyrrolidine-2-carboxamide(10) was prepared according to the general procedure A as yellow oil(9%). ¹H NMR (300 MHz, CDCl₃) δ 7.43 (dd, J=1.79, 7.44 Hz, 1H),7.28-7.34 (m, 2H), 7.12-7.25 (m, 3H), 6.87 (s, 2H), 3.90-3.96 (m, 2H),3.75 (s, 3H), 3.58-3.69 (m, 2H), 3.28 (s, 2H), 3.09 (dd, J=4.14, 7.54Hz, 1H), 2.68 (d, J=4.90 Hz, 3H), 2.43-2.59 (m, 1H), 2.03-2.14 (m, 2H).¹³C NMR (75 MHz, CDCl₃) δ 173.3, 160.5, 134.4, 133.9, 131.4, 131.0,129.6, 129.0, 127.1, 114.6, 66.2, 55.6, 55.2, 54.9, 54.8, 53.6, 42.0,33.5, 11.8. MS (ESI) [M]⁺388.3.

(2S,4S)-1-[(2-Chlorophenyl)methyl]-N-ethyl-4-{[(4-methoxyphenyl)methyl]amino}pyrrolidine-2-carboxamide(11) was prepared according to the general procedure A as yellow oil(32%). ¹H NMR (300 MHz, CDCl₃) δ 7.41 (br. s., 1H), 7.30-7.38 (m, 2H),7.14-7.25 (m, 4H), 6.84 (d, J=8.67 Hz, 1H), 3.83-3.91 (m, 1H), 3.79 (s,3H), 3.58-3.73 (m, 3H), 3.33 (td, J=2.80, 5.89 Hz, 1H), 3.19-3.28 (m,1H), 3.10-3.19 (m, 2H), 3.00 (d, J=9.98 Hz, 1H), 2.62 (dd, J=5.56, 10.08Hz, 1H), 2.49 (ddd, J=6.40, 9.84, 13.70 Hz, 1H), 1.87-1.97 (m, 1H), 1.02(t, J=14.70 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 173.9, 158.9, 135.7,134.3, 131.1, 129.7, 129.5, 128.9, 126.9, 113.9, 67.1, 59.5, 57.4, 56.2,55.3, 51.1, 37.0, 33.7, 14.6. MS (ESI) [M]⁺402.2.

(2S,4S)-1-[(2-Chlorophenyl)methyl]-4-{[(4-methoxyphenyl)methyl]amino}-N-propylpyrrolidine-2-carboxamide(12) was prepared according to the general procedure A as yellow oil(48%). ¹H NMR (300 MHz, CDCl₃) δ 7.82 (t, J=5.27 Hz, 1H), 7.29-7.50 (m,4H), 7.24 (d, J=8.67 Hz, 2H), 6.85 (d, J=8.67 Hz, 2H), 4.26-4.46 (m,3H), 4.14 (d, J=13.00 Hz, 2H), 3.94-4.03 (m, 1H), 3.78 (s, 3H), 3.46 (d,J=5.09 Hz, 2H), 3.07-3.22 (m, 2H), 2.88-3.02 (m, 1H), 2.27-2.40 (m, 1H),1.42-1.54 (m, 2H), 0.87 (t, J=7.44 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃) δ174.0, 158.9, 135.7, 134.1, 131.0, 129.7, 129.6, 128.8, 126.9, 113.9,67.1, 59.2, 57.2, 56.1, 55.3, 51.0, 40.7, 36.8, 22.7, 11.4. MS (ESI)[M]⁺416.5.

(2S,4S)—N-Butyl-1-[(2-chlorophenyl)methyl]-4-{[(4-methoxyphenyl)methyl]amino}pyrrolidine-2-carboxamide(13) was prepared according to the general procedure A as yellow oil(12%). ¹H NMR (300 MHz, CDCl₃) δ 7.74 (br. s., 1H), 7.47 (d, J=7.72 Hz,1H), 7.39-7.45 (m, 2H), 7.34 (dd, J=2.07, 7.16 Hz, 1H), 7.24 (d, J=8.67Hz, 2H), 6.87 (d, J=8.29 Hz, 2H), 4.34-4.56 (m, 3H), 4.19 (d, J=13.37Hz, 2H), 3.99-4.07 (m, 1H), 3.78 (s, 3H), 3.56 (d, J=3.20 Hz, 2H),3.13-3.27 (m, 2H), 2.94-3.08 (m, 1H), 2.33-2.46 (m, 1H), 1.38-1.50 (m,2H), 1.29 (dd, J=6.97, 14.88 Hz, 2H), 0.90 (t, J=7.25 Hz, 3H). ¹³C NMR(75 MHz, CDCl₃) δ 173.2, 159.8, 135.1, 133.9, 130.8, 130.6, 129.6,128.8, 127.0, 114.3, 66.7, 57.2, 55.8, 55.5, 55.2, 49.6, 39.0, 34.8,31.4, 20.0, 13.6. MS (ESI) [M]⁺430.1.

(2S,4S)—N—(Butan-2-yl)-1-[(2-chlorophenyl)methyl]-4-{[(4-methoxyphenyl)methyl]amino}pyrrolidine-2-carboxamide (14) was prepared according to the generalprocedure A as colorless oil (74%). ¹H NMR (300 MHz, CDCl₃) δ 7.29-7.39(m, 3H), 7.16-7.25 (m, 4H), 6.83 (d, J=8.67 Hz, 1H), 3.87-3.94 (m, 1H),3.74-3.84 (m, 5H), 3.61-3.72 (m, 3H), 3.20-3.33 (m, 2H), 2.97 (td,J=1.62, 10.13 Hz, 1H), 2.60 (ddd, J=3.01, 5.60, 10.03 Hz, 1H), 2.45-2.54(m, 1H), 1.87-1.96 (m, 1H), 1.76 (br. s., 1H), 1.29-1.39 (m, 2H), 1.02(d, J=6.59 Hz, 2H), 0.94 (d, J=6.59 Hz, 2H), 0.74-0.85 (m, 5H). ¹³C NMR(75 MHz, CDCl₃) δ 173.4, 173.4, 158.7, 158.7, 135.9, 134.3, 134.2,131.9, 131.2, 130.9, 129.8, 129.8, 129.4, 128.9, 128.8, 126.9, 126.9,113.8, 113.8, 67.3, 67.2, 59.7, 59.6, 57.7, 57.5, 56.2, 56.1, 55.3,51.3, 46.0, 45.8, 37.4, 37.2, 29.6, 20.1, 10.5, 10.4. MS (ESI)[M]⁺430.4.

(2S,4S)—N-tert-Butyl-1-[(2-chlorophenyl)methyl]-4-{[(4-methoxyphenyl)methyl]amino}pyrrolidine-2-carboxamide(15) was prepared according to the general procedure A as colorless oil(88%). ¹H NMR (300 MHz, CDCl₃) δ 7.30-7.39 (m, 2H), 7.15-7.25 (m, 5H),6.82-6.87 (m, 2H), 3.76-3.82 (m, 5H), 3.60-3.75 (m, 2H), 3.28-3.36 (m,1H), 3.14 (dd, J=5.27, 9.80 Hz, 1H), 3.06 (d, J=10.17 Hz, 1H), 2.64 (dd,J=5.65, 9.98 Hz, 1H), 2.47 (ddd, J=6.40, 9.80, 13.56 Hz, 1H), 1.89 (td,J=3.67, 13.56 Hz, 1H), 1.18 (s, 9H). ¹³C NMR (75 MHz, CDCl₃) δ 173.5,158.8, 136.0, 134.4, 131.0, 129.7, 129.4, 128.9, 127.0, 113.9, 67.8,59.9, 57.6, 56.2, 55.3, 51.2, 50.2, 37.2, 28.5. MS (ESI) [M]⁺430.0.

(2S,4S)-1-[(2-Chlorophenyl)methyl]-4-{[(4-methoxyphenyl)methyl]amino}-N-pentylpyrrolidine-2-carboxamide(16) was prepared according to the general procedure A as yellow oil(25%). ¹H NMR (300 MHz, CDCl₃) δ 7.32-7.43 (m, 3H), 7.17-7.24 (m, 4H),6.81-6.86 (m, 2H), 3.84-3.91 (m, 1H), 3.78 (s, 3H), 3.64-3.74 (m, 3H),3.31-3.38 (m, 1H), 3.25 (dd, J=5.65, 9.80 Hz, 1H), 2.99-3.16 (m, 3H),2.61 (dd, J=5.56, 10.27 Hz, 1H), 2.48 (dd, J=2.83, 6.78 Hz, 1H), 1.92(d, J=18.08 Hz, 1H), 1.31-1.43 (m, 2H), 1.16-1.30 (m, 4H), 0.84 (t,J=6.97 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 173.9, 158.9, 135.7, 134.1,130.9, 129.7, 129.6, 128.8, 126.9, 113.9, 67.1, 59.2, 57.2, 56.1, 55.3,51.0, 38.9, 36.8, 29.1, 29.1, 22.3, 13.9. MS (ESI) [M]⁺444.4.

(2S,4S)-1-[(2-Chlorophenyl)methyl]-4-{[(4-methoxyphenyl)methyl]amino}-N-(3-methylbutyl)pyrrolidine-2-carboxamide(17) was prepared according to the general procedure A as colorless oil(71%). ¹H NMR (300 MHz, CDCl₃) δ 7.23-7.34 (m, 3H), 7.10-7.18 (m, 4H),6.76 (d, J=8.67 Hz, 2H), 3.77-3.83 (m, 1H), 3.71 (s, 3H), 3.57-3.67 (m,3H), 3.27 (td, J=2.87, 5.93 Hz, 1H), 3.17 (dd, J=5.46, 9.80 Hz, 1H),3.05 (dt, J=7.72, 12.90 Hz, 2H), 2.92-2.98 (m, 1H), 2.54 (dd, J=5.65,10.17 Hz, 1H), 2.42 (ddd, J=6.59, 9.80, 13.75 Hz, 1H), 1.80-1.89 (m,1H), 1.44 (td, J=6.69, 13.37 Hz, 1H), 1.16-1.23 (m, 2H), 0.77 (dd,J=3.39, 6.59 Hz, 6H). ¹³C NMR (75 MHz, CDCl₃) δ 172.9, 157.9, 134.7,133.1, 130.0, 128.7, 128.6, 127.8, 125.9, 112.9, 66.1, 58.2, 56.2, 55.1,54.2, 49.9, 37.3, 36.2, 35.8, 24.8, 21.4. MS (ESI) [M]⁺444.7.

(2S,4S)-1-[(2-Chlorophenyl)methyl]-4-{[(4-methoxyphenyl)methyl]amino}-N-hexylpyrrolidine-2-carboxamide(18) was prepared according to the general procedure A as colorless oil(49%). ¹H NMR (300 MHz, CDCl₃) δ 7.45 (t, J=5.65 Hz, 1H), 7.31-7.38 (m,2H), 7.16-7.24 (m, 4H), 6.81-6.85 (m, 2H), 3.84-3.91 (m, 1H), 3.79 (s,3H), 3.58-3.72 (m, 3H), 3.31 (td, J=2.76, 5.98 Hz, 1H), 3.25 (dd,J=5.56, 9.89 Hz, 1H), 3.11 (dt, J=7.06, 12.86 Hz, 2H), 3.00 (d, J=10.17Hz, 1H), 2.60 (dd, J=5.56, 10.08 Hz, 1H), 2.49 (ddd, J=6.50, 9.84, 13.61Hz, 1H), 1.90 (td, J=3.84, 13.61 Hz, 2H), 1.31-1.41 (m, 2H), 1.17-1.29(m, 6H), 0.85 (t, J=13.40 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 174.0,158.8, 135.8, 134.2, 131.8, 131.0, 129.7, 129.4, 128.8, 126.9, 113.9,67.2, 59.6, 57.4, 56.2, 55.2, 51.2, 38.9, 37.2, 31.5, 29.4, 26.6, 22.5,14.0. MS (ESI) [M]⁺458.0.

(2S,4S)-1-[(2-Chlorophenyl)methyl]-N-decyl-4-{[(4-methoxyphenyl)methyl]amino}pyrrolidine-2-carboxamide(19) was prepared according to the general procedure A as colorless oil(58%). ¹H NMR (300 MHz, CDCl₃) δ 7.42 (t, J=5.75 Hz, 1H), 7.33-7.37 (m,2H), 7.16-7.25 (m, 5H), 6.83 (d, J=8.67 Hz, 1H), 3.84-3.91 (m, 1H), 3.78(s, 3H), 3.62-3.75 (m, 4H), 3.33-3.40 (m, 1H), 3.25 (dd, J=5.65, 9.61Hz, 1H), 2.90-3.16 (m, 6H), 2.61 (dd, J=5.65, 10.17 Hz, 1H), 2.49 (ddd,J=6.59, 9.61, 13.75 Hz, 1H), 1.89-1.97 (m, 1H), 1.16-1.42 (m, 18H),0.85-0.90 (m, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 173.9, 159.0, 135.7, 134.1,130.9, 129.7, 129.7, 128.8, 127.0, 114.0, 67.1, 59.1, 57.1, 56.1, 55.3,50.9, 39.0, 36.7, 31.9, 29.6, 29.5, 29.5, 29.3, 27.0, 22.7, 14.1. MS(ESI) [M]⁺514.8.

(2S,4S)-1-[(2-Chlorophenyl)methyl]-4-{[(4-methoxyphenyl)methyl]amino}-N—[2-(morpholin-4-yl)ethyl]pyrrolidine-2-carboxamide(20) was prepared according to the general procedure A as colorless oil(18%). ¹H NMR (300 MHz, CDCl₃) δ 7.52 (t, J=5.27 Hz, 1H), 7.34-7.38 (m,2H), 7.18-7.25 (m, 4H), 6.84 (d, J=8.67 Hz, 2H), 3.84-3.91 (m, 1H),3.74-3.78 (m, 4H), 3.68-3.73 (m, 2H), 3.59-3.65 (m, 4H), 3.42 (t, J=7.72Hz, 1H), 3.22-3.37 (m, 4H), 2.35-2.45 (m, 7H), 2.13-2.21 (m, J=1.00,1.00 Hz, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 173.5, 159.2, 135.6, 134.2,130.9, 129.8, 129.7, 128.9, 126.9, 114.0, 66.8, 66.4, 57.4, 57.3, 55.4,55.2, 53.4, 51.4, 36.7, 35.3, 30.9. MS (ESI) [M]⁺487.6.

(2S,4S)-1-[(2-Chlorophenyl)methyl]-N-(2-cyclohexylethyl)-4-{[(4-methoxyphenyl)methyl]amino}pyrrolidine-2-carboxamide (21) was prepared according to thegeneral procedure A as colorless oil (53%). ¹H NMR (300 MHz, CDCl₃) δ7.32-7.41 (m, 3H), 7.15-7.25 (m, 4H), 6.84 (d, J=8.67 Hz, 2H), 3.83-3.90(m, 1H), 3.79 (s, 3H), 3.59-3.73 (m, 3H), 3.32 (td, J=2.80, 5.89 Hz,1H), 3.25 (dd, J=5.56, 9.70 Hz, 1H), 3.08-3.19 (m, 2H), 3.02 (d, J=10.17Hz, 1H), 2.60 (dd, J=5.65, 9.98 Hz, 1H), 2.49 (ddd, J=6.50, 9.80, 13.66Hz, 1H), 1.86-1.95 (m, 1H), 1.56-1.70 (m, 5H), 1.08-1.30 (m, 6H),0.76-0.93 (m, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 173.9, 158.8, 135.8, 134.1,130.9, 129.7, 129.5, 128.8, 126.9, 113.9, 67.2, 59.4, 57.3, 56.2, 55.2,51.1, 37.0, 36.8, 36.7, 35.3, 33.1, 33.1, 30.9, 26.5, 26.2. MS (ESI)[M]⁺484.4.

(2S,4S)-1-[(2-Chlorophenyl)methyl]-N,N-diethyl-4-{[(4-methoxyphenyl)methyl]amino}pyrrolidine-2-carboxamide(22) was prepared according to the general procedure A as colorless oil(11%). ¹H NMR (300 MHz, CDCl₃) δ 7.78 (br. s., 1H), 7.46 (dd, J=2.26,6.97 Hz, 1H), 7.33-7.38 (m, 1H), 7.16-7.25 (m, 4H), 6.80-6.88 (m, 2H),3.83-3.91 (m, 1H), 3.79 (s, 3H), 3.64-3.76 (m, 3H), 3.16-3.36 (m, 4H),3.03 (d, J=9.61 Hz, 1H), 2.43-2.61 (m, 7H), 1.79-2.05 (m, 3H), 0.96 (t,J=7.06 Hz, 6H). ¹³C NMR (75 MHz, CDCl₃) δ 174.2, 158.8, 135.8, 134.0,130.8, 129.5, 129.4, 128.6, 126.9, 113.9, 67.2, 56.8, 56.1, 55.3, 51.5,51.2, 46.6, 37.3, 36.2, 11.1. MS (ESI) [M]⁺473.5.

(2S,4S)—N-Benzyl-1-[(2-chlorophenyl)methyl]-4-{[(4-methoxyphenyl)methyl]amino}pyrrolidine-2-carboxamide(23) was prepared according to the general procedure A as yellow oil(44%). ¹H NMR (300 MHz, CDCl₃) δ 7.82 (t, J=5.75 Hz, 1H), 7.22-7.27 (m,5H), 7.10-7.21 (m, 6H), 6.77-6.83 (m, 2H), 4.32 (dd, J=5.93, 8.95 Hz,2H), 3.87 (d, J=13.19 Hz, 1H), 3.77 (s, 3H), 3.58-3.71 (m, 3H),3.34-3.41 (m, 1H), 3.31 (dd, J=5.56, 9.70 Hz, 1H), 2.98 (d, J=10.36 Hz,1H), 2.60 (dd, J=5.46, 10.36 Hz, 1H), 2.49 (ddd, J=6.78, 9.70, 13.85 Hz,1H), 1.92-2.02 (m, 1H). ¹³C NMR (75 MHz, CDCl₃) δ 173.9, 159.1, 138.3,135.4, 134.1, 131.0, 129.7, 129.7, 128.8, 128.6, 127.8, 127.3, 126.9,114.0, 67.0, 58.7, 56.9, 56.0, 55.3, 50.7, 43.1, 36.3. MS (ESI)[M]⁺464.4.

(2S,4S)-1-[(2-Chlorophenyl)methyl]-4-{[(4-methoxyphenyl)methyl]amino}-N-(2-phenylethyl)pyrrolidine-2-carboxamide(24) was prepared according to the general procedure A as yellow oil(59%). ¹H NMR (300 MHz, CDCl₃) δ 7.52 (t, J=5.84 Hz, 1H), 7.32-7.37 (m,1H), 7.09-7.24 (m, 11H), 6.83 (d, J=8.67 Hz, 1H), 3.73-3.81 (m, 4H),3.63-3.67 (m, 1H), 3.56-3.62 (m, 2H), 3.43-3.55 (m, 1H), 3.30-3.42 (m,1H), 3.19-3.28 (m, 2H), 2.92 (d, J=10.17 Hz, 1H), 2.67-2.76 (m, 2H),2.42-2.55 (m, 2H), 1.96 (br. s., 1H), 1.81 (m, 1H). ¹³C NMR (75 MHz,CDCl₃) δ 174.1, 158.7, 138.9, 135.8, 134.1, 132.0, 130.7, 129.6, 129.4,128.7, 128.6, 128.5, 126.9, 126.4, 113.9, 67.3, 59.5, 57.2, 56.0, 55.3,51.2, 39.8, 37.3, 35.5. MS (ESI) [M]⁺478.4.

(2S,4S)-1-[(2-Chlorophenyl)methyl]-4-{[(4-methoxyphenyl)methyl]amino}-N-(3-phenylpropyl)pyrrolidine-2-carboxamide(25) was prepared according to the general procedure A as colorless oil(70%). ¹H NMR (300 MHz, CDCl₃) δ 7.44 (t, J=5.37 Hz, 1H), 7.13-7.36 (m,10H), 7.09 (d, J=6.78 Hz, 1H), 6.80 (d, J=8.67 Hz, 2H), 3.83-3.90 (m,1H), 3.75 (s, 3H), 3.66-3.73 (m, 3H), 3.34-3.41 (m, 1H), 3.25 (dd,J=5.56, 9.70 Hz, 1H), 3.11-3.20 (m, 2H), 3.05 (d, J=10.55 Hz, 1H),2.43-2.64 (m, 4H), 1.96 (d, J=3.20 Hz, 1H), 1.65-1.77 (m, 2H). ¹³C NMR(75 MHz, CDCl₃) δ 173.9, 159.1, 141.5, 135.6, 134.1, 130.9, 129.8,129.7, 128.9, 128.4, 128.3, 127.0, 125.9, 114.0, 67.0, 58.9, 56.9, 56.0,55.2, 50.8, 38.6, 36.5, 33.2, 31.1. MS (ESI) [M]⁺492.7.

(2S,4S)-1-[(2-Chlorophenyl)methyl]-N—[2-(4-methoxyphenyl)ethyl]-4-{[(4-methoxyphenyl)methyl]amino}pyrrolidine-2-carboxamide (26) was prepared according tothe general procedure A as colorless oil (20%). ¹H NMR (300 MHz, CDCl₃)δ 7.40 (t, J=5.65 Hz, 1H), 7.32-7.36 (m, 1H), 7.15-7.22 (m, 4H),7.10-7.14 (m, 1H), 7.05 (d, J=8.67 Hz, 2H), 6.85 (d, J=8.67 Hz, 2H),6.77 (d, J=8.48 Hz, 2H), 3.78 (s, 3H), 3.75 (s, 3H), 3.70-3.73 (m, 1H),3.65 (d, J=9.04 Hz, 2H), 3.42-3.52 (m, 1H), 3.37 (dd, J=5.65, 9.61 Hz,1H), 3.23-3.32 (m, 1H), 3.10-3.22 (m, 2H), 2.67 (dt, J=3.30, 7.02 Hz,2H), 2.25 (t, J=8.01 Hz, 1H), 2.01-2.16 (m, 2H), 1.40-1.68 (m, 2H). ¹³CNMR (75 MHz, CDCl₃) δ 173.8, 158.8, 158.2, 135.7, 134.2, 132.1, 131.0,130.9, 129.6, 129.6, 129.2, 128.8, 126.9, 114.0, 113.9, 66.7, 60.1,57.8, 56.1, 55.3, 55.2, 52.1, 40.0, 37.9, 34.8, 30.9. MS (ESI)[M]⁺508.6.

(2S,4S)—N—[2-(4-Chlorophenyl)ethyl]-1-[(2-chlorophenyl)methyl]-4-{[(4-methoxyphenyl)methyl]amino}pyrrolidine-2-carboxamide(27) was prepared according to the general procedure A as colorless oil(55%). ¹H NMR (300 MHz, CDCl₃) δ 7.46 (t, J=5.93 Hz, 1H), 7.27-7.31 (m,1H), 7.08-7.25 (m, 7H), 7.00-7.04 (m, 2H), 6.81-6.86 (m, 2H), 3.75-3.80(m, OH), 3.73 (s, 3H), 3.68 (d, J=13.19 Hz, 2H), 3.53-3.60 (m, 1H),3.31-3.50 (m, 3H), 3.18 (dd, J=6.78, 9.04 Hz, 1H), 3.08 (d, J=10.93 Hz,1H), 2.69 (q, J=7.03 Hz, 2H), 2.42-2.55 (m, 2H), 1.92 (dt, J=3.20, 7.06Hz, 1H). ¹³C NMR (75 MHz, CDCl₃) δ 173.1, 159.8, 137.2, 134.9, 133.7,132.1, 130.6, 130.2, 130.0, 129.6, 128.7, 128.5, 126.9, 114.3, 67.1,57.3, 55.8, 55.2, 55.2, 49.7, 39.7, 34.9, 34.7. MS (ESI) [M]⁺512.4.

(2S,4S)-1-[(2-Chlorophenyl)methyl]-N—[2-(3,4-dimethoxyphenyl)ethyl]-4-{[(4-methoxyphenyl)methyl]amino}pyrrolidine-2-carboxamide(28) was prepared according to the general procedure A as white solid(61%). ¹H NMR (300 MHz, CDCl₃) δ 7.29-7.35 (m, 1H), 7.18-7.25 (m, 3H),7.09-7.15 (m, 2H), 6.82 (d, J=8.67 Hz, 2H), 6.68 (s, 3H), 3.63-3.88 (m,13H), 3.45-3.60 (m, 3H), 3.28-3.41 (m, 1H), 3.12-3.20 (m, 2H), 2.70 (qd,J=7.21, 14.93 Hz, 2H), 2.43-2.58 (m, 2H), 1.99 (ddd, J=3.49, 6.26, 13.99Hz, 1H). ¹³C NMR (75 MHz, CDCl₃) δ 172.6, 160.3, 148.9, 147.6, 134.6,133.6, 131.2, 131.1, 130.4, 129.5, 128.7, 126.9, 120.6, 114.5, 111.8,111.1, 66.8, 56.0, 55.8, 55.2, 55.0, 54.9, 48.9, 40.1, 34.8, 33.8. MS(ESI) [M]⁺538.4.

(2S,4S)-1-[(2-Chlorophenyl)methyl]-N—{2-[4-(dimethylamino)phenyl]ethyl}-4-{[(4-methoxyphenyl)methyl]amino}pyrrolidine-2-carboxamide(29) was prepared according to the general procedure A as yellow oil(25%). ¹H NMR (300 MHz, CDCl₃) δ 7.30-7.34 (m, 1H), 7.15-7.25 (m, 5H),6.98 (d, J=8.67 Hz, 2H), 6.84 (d, J=8.67 Hz, 2H), 6.59 (d, J=8.48 Hz,2H), 3.62-3.81 (m, 7H), 3.33-3.45 (m, 3H), 3.20 (dd, J=5.46, 9.42 Hz,1H), 3.05 (d, J=10.36 Hz, 1H), 2.86 (s, 6H), 2.53-2.69 (m, 3H),2.40-2.51 (m, 1H), 1.91 (d, J=13.56 Hz, 1H). ¹³C NMR (75 MHz, CDCl₃) δ173.7, 159.3, 149.4, 135.4, 133.9, 130.8, 130.1, 129.5, 129.2, 128.6,126.9, 126.5, 114.1, 112.9, 66.8, 58.2, 56.1, 55.8, 55.3, 50.3, 40.7,40.3, 35.8, 34.4. MS (ESI) [M]⁺521.8.

(2S,4S)-1-[(2-Chlorophenyl)methyl]-N—[2-(4-acetamidophenyl)ethyl]-4-{[(4-methoxyphenyl)methyl]amino}pyrrolidine-2-carboxamide(30) was prepared according to the general procedure A as colorlessliquid (50%). ¹H NMR (300 MHz, CDCl₃) δ 8.44 (br. s., 1H), 7.67 (t,J=5.75 Hz, 1H), 7.37 (dd, J=1.60, 7.44 Hz, 1H), 7.23-7.31 (m, 6H),7.11-7.21 (m, 2H), 7.03 (d, J=8.29 Hz, 2H), 6.86 (d, J=8.67 Hz, 2H),3.84 (s, 2H), 3.76 (s, 3H), 3.62-3.74 (m, 2H), 3.39-3.48 (m, 2H), 3.34(td, J=6.31, 12.62 Hz, 1H), 3.11-3.23 (m, 2H), 2.66-2.75 (m, 2H), 2.54(dd, J=5.56, 11.02 Hz, 1H), 2.28-2.41 (m, 1H), 2.07 (s, 3H), 1.76 (d,J=14.32 Hz, 1H). ¹³C NMR (75 MHz, CDCl₃) δ 173.1, 168.9, 160.1, 136.1,134.5, 134.5, 133.5, 130.9, 130.5, 129.2, 129.0, 128.5, 126.8, 120.6,114.3, 65.6, 55.0, 54.8, 54.4, 53.5, 48.7, 39.8, 34.4, 33.7, 23.9. MS(ESI) [M]⁺535.6.

(2S,4S)-1-[(2-Chlorophenyl)methyl]-4-{[(4-methoxyphenyl)methyl]amino}-N—{2-[4-(methylcarbamoyl)phenyl]ethyl}pyrrolidine-2-carboxamide(31) was prepared according to the general procedure A as colorless oil(28%). ¹H NMR (300 MHz, CDCl₃) δ 7.75 (t, J=5.84 Hz, 1H), 7.53 (d,J=8.10 Hz, 2H), 7.37 (dd, J=2.17, 6.88 Hz, 1H), 7.22 (d, J=8.67 Hz, 2H),7.09-7.18 (m, 4H), 6.82 (d, J=8.48 Hz, 3H), 3.86 (d, J=2.45 Hz, 2H),3.71-3.77 (m, 3H), 3.57-3.68 (m, 2H), 3.33-3.54 (m, 3H), 3.12-3.23 (m,2H), 3.06 (q, J=7.41 Hz, 1H), 2.89 (d, J=4.71 Hz, 3H), 2.74-2.83 (m,2H), 2.38-2.55 (m, 2H), 1.94 (dd, J=3.58, 14.13 Hz, 1H), 1.34-1.45 (m,5H). ¹³C NMR (75 MHz, CDCl₃) δ 173.0, 168.3, 160.4, 142.5, 134.7, 133.6,132.7, 131.3, 130.5, 129.5, 128.8, 128.7, 127.1, 114.5, 66.5, 55.2,54.9, 54.7, 53.7, 48.9, 42.0, 39.8, 35.0, 33.8, 26.7, 11.8. MS (ESI)[M]⁺535.5.

(2S,4S)—N—[2-(4-tert-Butylphenyl)ethyl]-1-[(2-chlorophenyl)methyl]-4-{[(4-methoxyphenyl)methyl]amino}pyrrolidine-2-carboxamide(32) was prepared according to the general procedure A as colorless oil(36%). ¹H NMR (300 MHz, CDCl₃) δ 7.51 (br. s., 1H), 7.31-7.36 (m, 1H),7.14-7.25 (m, 8H), 7.05 (d, J=8.29 Hz, 2H), 6.83 (d, J=8.67 Hz, 2H),3.75-3.83 (m, 5H), 3.57-3.67 (m, 3H), 3.40-3.50 (m, 1H), 3.30-3.39 (m,1H), 3.20-3.26 (m, 2H), 2.92 (d, J=10.17 Hz, 1H), 2.67 (q, J=7.03 Hz,2H), 2.41-2.56 (m, 2H), 1.80-1.89 (m, 1H), 1.26 (s, 9H). ¹³C NMR (75MHz, CDCl₃) δ 174.1, 158.7, 149.2, 135.8, 135.8, 134.1, 132.1, 130.9,129.6, 129.3, 128.7, 128.3, 126.9, 125.4, 113.8, 67.2, 59.6, 57.2, 56.1,55.2, 51.2, 39.9, 37.3, 35.1, 34.3, 31.3. MS (ESI) [M]⁺534.3.

(2S,4S)-1-[(2-Chlorophenyl)methyl]-N—[2-(4-methoxyphenyl)ethyl]-4-{[(4-nitrophenyl)methyl]amino}pyrrolidine-2-carboxamide (33) was prepared according tothe general procedure A as yellow oil (22%). ¹H NMR (300 MHz, CDCl₃) δ8.06 (d, J=8.67 Hz, 2H), 7.45-7.51 (m, 1H), 7.31-7.36 (m, 1H), 7.27 (s,1H), 7.13-7.26 (m, 6H), 6.82-6.89 (m, 2H), 3.79 (s, 3H), 3.62-3.74 (m,4H), 3.34-3.53 (m, 3H), 3.11-3.22 (m, 2H), 2.83 (dt, J=2.92, 6.92 Hz,2H), 2.22-2.33 (m, 1H), 2.08 (t, J=7.54 Hz, 2H), 1.46-1.72 (m, 2H). ¹³CNMR (75 MHz, CDCl₃) δ 174.1, 158.9, 146.7, 146.7, 135.5, 134.1, 130.9,129.7, 129.5, 129.2, 129.0, 128.8, 127.0, 123.7, 113.9, 66.6, 60.1,58.1, 55.9, 55.3, 52.0, 39.2, 37.8, 35.6. MS (ESI) [M]⁺523.7.

(2S,4S)-1-[(2-Chlorophenyl)methyl]-N—[2-(3,4-difluorophenyl)ethyl]-4-{[(4-methoxyphenyl)methyl]amino}pyrrolidine-2-carboxamide(34) was prepared according to the general procedure A as white solid(62%). ¹H NMR (300 MHz, CDCl₃) δ 7.55 (t, J=6.03 Hz, 1H), 7.29 (d,J=2.26 Hz, 1H), 7.11-7.25 (m, 5H), 6.88-6.96 (m, 2H), 6.75-6.86 (m, 3H),3.72-3.79 (m, 4H), 3.54-3.71 (m, 3H), 3.30-3.47 (m, 3H), 3.19 (dd,J=6.69, 9.14 Hz, 1H), 3.07 (d, J=10.93 Hz, 1H), 2.67 (q, J=6.91 Hz, 2H),2.43-2.56 (m, 2H), 1.94 (dd, J=3.11, 6.50 Hz, 1H). ¹³C NMR (75 MHz,CDCl₃) δ 173.2, 159.7, 135.8, 135.0, 133.7, 130.5, 130.2, 129.6, 128.7,126.9, 124.5, 124.5, 124.4, 124.4, 117.5, 117.2, 117.1, 116.9, 114.2,67.2, 57.6, 56.0, 55.3, 55.2, 49.8, 39.6, 38.6, 35.1, 34.5. MS (ESI)[M]⁺514.5.

(2S,4S)-1-[(2-Chlorophenyl)methyl]-N—[2-(4-fluorophenyl)ethyl]-4-{[(4-methoxyphenyl)methyl]amino}pyrrolidine-2-carboxamide(35) was prepared according to the general procedure A as colorless oil(74%). ¹H NMR (300 MHz, CDCl₃) δ 7.38 (t, J=5.84 Hz, 1H), 7.16-7.26 (m,4H), 7.01-7.12 (m, 4H), 6.77-6.86 (m, 4H), 3.73-3.87 (m, 2H), 3.70 (s,3H), 3.60-3.67 (m, 1H), 3.47-3.58 (m, 3H), 3.24-3.37 (m, 1H), 3.10-3.21(m, 2H), 2.63-2.81 (m, 2H), 2.44-2.56 (m, 2H), 1.93-2.04 (m, 1H). ¹³CNMR (75 MHz, CDCl₃) δ 172.3, 160.5, 134.4, 134.3, 134.3, 133.5, 131.4,130.0, 129.9, 129.5, 128.6, 126.9, 121.8, 115.3, 115.0, 114.5, 67.1,55.8, 55.2, 54.9, 54.6, 48.7, 40.1, 34.4, 33.4. MS (ESI) [M]⁺496.7.

(2S,4S)-1-[(2-Chlorophenyl)methyl]-4-{[(4-methoxyphenyl)methyl]amino}-N—{2-[4-(trifluoromethyl)phenyl]ethyl}pyrrolidine-2-carboxamide(36) was prepared according to the general procedure A as yellow oil(69%). ¹H NMR (300 MHz, CDCl₃) δ 7.54 (t, J=5.84 Hz, 1H), 7.43 (d,J=8.10 Hz, 2H), 7.32 (dd, J=1.88, 7.35 Hz, 1H), 7.14-7.24 (m, 7H),6.81-6.86 (m, 2H), 3.73-3.80 (m, 4H), 3.57-3.70 (m, 3H), 3.38-3.48 (m,2H), 3.29-3.36 (m, 1H), 3.23 (dd, J=5.84, 9.80 Hz, 1H), 3.00 (s, 1H),2.71-2.81 (m, 2H), 2.42-2.58 (m, 2H), 1.80-1.90 (m, 1H). ¹³C NMR (75MHz, CDCl₃) δ 174.0, 159.0, 143.0, 135.5, 134.0, 130.6, 129.6, 129.0,128.8, 126.9, 125.4, 125.3, 114.0, 67.1, 59.0, 57.0, 56.0, 55.2, 50.9,39.5, 36.7, 35.4. MS (ESI) [M]⁺546.7.

(2S,4S)-1-[(2-Chlorophenyl)methyl]-4-{[(4-methoxyphenyl)methyl]amino}-N—[2-(pyridin-4-yl)ethyl]pyrrolidine-2-carboxamide(37) was prepared according to the general procedure A as white solid(73%). ¹H NMR (300 MHz, CDCl₃) δ 8.34 (d, J=5.84 Hz, 2H), 7.65 (t,J=5.93 Hz, 1H), 7.24-7.33 (m, 3H), 7.20 (d, J=8.67 Hz, 2H), 7.10-7.15(m, 2H), 7.05 (d, J=6.03 Hz, 2H), 6.82 (d, J=8.67 Hz, 2H), 3.75-3.86 (m,2H), 3.67 (br. s., 5H), 3.50 (quin, J=6.83 Hz, 2H), 3.35-3.44 (m, 1H),3.10-3.20 (m, 2H), 2.76 (dt, J=2.83, 7.06 Hz, 2H), 2.44-2.57 (m, 2H),1.93 (ddd, J=2.73, 7.16, 14.22 Hz, 1H). ¹³C NMR (75 MHz, CDCl₃) δ 172.7,160.3, 149.4, 148.3, 134.5, 133.6, 131.2, 130.2, 129.6, 128.7, 126.9,124.2, 114.5, 67.1, 56.2, 55.2, 54.8, 49.1, 38.9, 34.6, 34.0. MS (ESI)[M]⁺479.3.

(2S,4S)—N—[2-(4-Acetylphenyl)ethyl]-1-[(2-chlorophenyl)methyl]-4-{[(4-methoxyphenyl)methyl]amino}pyrrolidine-2-carboxamide(38) was prepared according to the general procedure A as colorlessliquid (35%). ¹H NMR (300 MHz, CDCl₃) δ 7.65-7.72 (m, 2H), 7.35-7.42 (m,1H), 7.18-7.24 (m, 3H), 7.05-7.16 (m, 4H), 6.83 (d, J=8.67 Hz, 2H),3.78-3.89 (m, 2H), 3.66-3.74 (m, 4H), 3.48-3.61 (m, 3H), 3.37 (d, J=5.65Hz, 1H), 3.18 (d, J=8.10 Hz, 2H), 2.74-2.87 (m, 2H), 2.43-2.60 (m, 2H),2.18 (d, J=5.84 Hz, 3H), 1.93-2.06 (m, 1H). ¹³C NMR (75 MHz, CDCl₃) δ172.4, 160.5, 157.6, 140.4, 136.6, 134.4, 133.7, 131.5, 130.5, 129.5,128.7, 128.6, 127.0, 126.7, 121.7, 114.6, 66.9, 55.5, 55.2, 54.7, 54.2,48.6, 40.0, 35.1, 33.4, 14.8. MS (ESI) [M]⁺519.5.

(2S,4S)-1-[(2-Chlorophenyl)methyl]-N—[2-(4-methanesulfonylphenyl)ethyl]-4-{[(4-methoxyphenyl)methyl]amino}pyrrolidine-2-carboxamide(39) was prepared according to the general procedure A as colorless oil(55%). ¹H NMR (300 MHz, CDCl₃) δ 7.75 (d, J=8.29 Hz, 2H), 7.64 (t,J=5.93 Hz, 1H), 7.25-7.35 (m, 5H), 7.16-7.22 (m, 4H), 6.81-6.86 (m, 2H),3.76 (s, 3H), 3.59-3.74 (m, 4H), 3.42 (q, J=6.97 Hz, 2H), 3.32-3.37 (m,1H), 3.23 (dd, J=5.84, 9.61 Hz, 1H), 3.01 (d, J=1.70 Hz, 1H), 2.98 (s,3H), 2.79 (qd, J=6.99, 14.46 Hz, 2H), 2.55 (dd, J=5.65, 10.55 Hz, 1H),2.41-2.50 (m, 1H), 1.81-1.91 (m, 1H). ¹³C NMR (75 MHz, CDCl₃) δ 174.0,159.2, 145.6, 138.7, 135.4, 133.9, 130.7, 129.9, 129.7, 129.6, 128.9,127.5, 127.0, 114.1, 67.0, 58.6, 56.7, 55.9, 55.3, 50.7, 44.5, 39.5,36.3, 35.5. MS (ESI) [M]⁺557.1.

(2S,4S)-1-[(2-Chlorophenyl)methyl]-N,N-diethyl-4-{[(4-methoxyphenyl)methyl]amino}pyrrolidine-2-carboxamide(40) was prepared according to the general procedure A as yellow oil(54%). ¹H NMR (300 MHz, CDCl₃) δ 7.37-7.44 (m, 3H), 7.31-7.35 (m, 1H),7.18-7.23 (m, 2H), 6.87-6.93 (m, 2H), 4.09 (d, J=2.07 Hz, 2H), 3.91-4.00(m, 3H), 3.82-3.86 (m, 1H), 3.81 (s, 3H), 3.39-3.51 (m, 2H), 3.18-3.29(m, 1H), 2.88-3.08 (m, 3H), 2.36-2.48 (m, 1H), 2.14 (d, J=14.69 Hz, 1H),1.04 (t, J=7.25 Hz, 3H), 0.97 (t, J=7.16 Hz, 3H). ¹³C NMR (75 MHz,CDCl₃) δ 174.0, 160.3, 135.1, 133.6, 131.3, 130.7, 129.5, 128.8, 127.1,123.1, 114.6, 59.0, 56.7, 56.4, 55.3, 54.3, 48.5, 42.4, 41.6, 33.7,15.0, 12.8. MS (ESI) [M]⁺430.4.

(2S,4S)-1-[(2-Chlorophenyl)methyl]-4-{[(4-methoxyphenyl)methyl]amino}-N,N-dipropylpyrrolidine-2-carboxamide(41) was prepared according to the general procedure A as colorless oil(63%). ¹H NMR (300 MHz, CDCl₃) δ 7.55 (dd, J=1.70, 7.54 Hz, 1H),7.13-7.34 (m, 6H), 6.85 (d, J=8.48 Hz, 2H), 3.73-3.92 (m, 7H), 3.70 (dd,J=4.52, 8.85 Hz, 1H), 3.44 (d, J=5.84 Hz, 1H), 2.99-3.35 (m, 6H), 2.81(dd, J=6.03, 9.61 Hz, 1H), 2.40 (td, J=8.34, 13.28 Hz, 1H), 1.80-1.89(m, 1H), 1.43-1.57 (m, 4H), 0.86 (dt, J=4.52, 7.44 Hz, 6H). ¹³C NMR (75MHz, CDCl₃) δ 173.4, 158.9, 136.4, 133.6, 130.8, 129.7, 129.2, 128.1,126.8, 113.9, 60.9, 58.3, 56.3, 55.3, 53.9, 50.7, 49.4, 48.3, 36.3,22.8, 20.9, 11.4, 11.2. MS (ESI) [M]⁺458.2.

(3S,5S)-1-[(2-Chlorophenyl)methyl]-N-[(4-methoxyphenyl)methyl]-5-(4-phenylpiperazine-1-carbonyl)pyrrolidin-3-amine(42) was prepared according to the general procedure A as colorless oil(23%). ¹H NMR (300 MHz, CDCl₃) δ 7.45 (dd, J=1.79, 7.44 Hz, 1H),7.27-7.39 (m, 5H), 7.14-7.25 (m, 2H), 6.84-6.95 (m, 5H), 3.90-3.99 (m,4H), 3.85 (dd, J=3.49, 9.14 Hz, 1H), 3.80 (s, 3H), 3.77 (s, 1H),3.54-3.71 (m, 3H), 3.46 (br. s., 1H), 3.35 (d, J=10.55 Hz, 1H),2.88-3.10 (m, 5H), 2.37-2.49 (m, 1H), 2.06 (d, J=14.13 Hz, 1H). ¹³C NMR(75 MHz, CDCl₃) δ 172.6, 159.7, 150.7, 135.4, 133.7, 130.9, 130.6,129.6, 129.3, 128.8, 127.0, 120.7, 116.6, 114.3, 57.5, 56.4, 55.3, 54.6,49.7, 49.6, 49.3, 45.6, 42.2, 34.7. MS (ESI) [M]⁺519.8.

(2S,4S)-1-[(2-Chlorophenyl)methyl]-N-ethyl-4-{[(4-(methylthio)phenyl)methyl]amino}pyrrolidine-2-carboxamide(43) was prepared according to the general procedure A as colorless oil(52%). ¹H NMR (300 MHz, CDCl₃) δ 7.45 (br. s., 1H), 7.37 (dd, J=3.67,5.56 Hz, 1H), 7.29-7.33 (m, 1H), 7.23 (dd, J=3.58, 5.84 Hz, 2H),7.16-7.21 (m, 4H), 3.83-3.90 (m, 1H), 3.63-3.73 (m, 3H), 3.29 (d, J=5.84Hz, 1H), 3.24 (dd, J=5.46, 9.98 Hz, 1H), 3.10-3.19 (m, 2H), 2.98 (d,J=9.98 Hz, 1H), 2.61 (dd, J=5.56, 10.08 Hz, 1H), 2.49-2.54 (m, 1H), 2.46(s, 3H), 1.91 (dd, J=2.73, 8.76 Hz, 3H), 1.02 (t, J=7.35 Hz, 3H). ¹³CNMR (75 MHz, CDCl₃) δ 173.5, 136.6, 136.3, 135.3, 133.8, 130.7, 129.3,128.5, 128.3, 126.5, 126.5, 66.6, 59.2, 57.1, 55.8, 50.8, 36.7, 33.2,15.6, 14.1. MS (ESI) [M]⁺418.3.

(2S,4S)-1-[(2-Chlorophenyl)methyl]-4-{[(4-(methylthio)phenyl)methyl]amino}-N-pentylpyrrolidine-2-carboxamide(44) was prepared according to the general procedure A as colorless oil(62%). ¹H NMR (300 MHz, CDCl₃) δ 7.47 (br. s., 1H), 7.31-7.39 (m, 2H),7.21-7.26 (m, 2H), 7.17-7.20 (m, 4H), 3.84-3.91 (m, 1H), 3.59-3.75 (m,3H), 3.30 (br. s., 1H), 3.25 (dd, J=5.46, 9.80 Hz, 1H), 3.10 (dt,J=6.97, 12.72 Hz, 2H), 2.99 (d, J=10.17 Hz, 1H), 2.60 (dd, J=5.46, 9.98Hz, 1H), 2.48-2.54 (m, 1H), 2.46 (s, 3H), 1.91 (d, J=10.36 Hz, 2H),1.32-1.43 (m, 2H), 1.17-1.29 (m, 4H), 0.84 (t, J=6.78 Hz, 3H). ¹³C NMR(75 MHz, CDCl₃) δ 174.0, 137.1, 136.7, 135.8, 134.2, 131.0, 129.7,128.9, 128.7, 126.9, 67.1, 59.6, 57.5, 56.3, 51.3, 38.9, 37.1, 29.2,29.1, 22.3, 16.1, 13.9. MS (ESI) [M]⁺460.3.

(2S,4S)-1-[(2-Chlorophenyl)methyl]-4-{[(4-(methylthio)phenyl)methyl]amino}-N-(2-phenylethyl)pyrrolidine-2-carboxamide(45) was prepared according to the general procedure A as colorless oil(46%). ¹H NMR (300 MHz, CDCl₃) δ 7.40 (br. s., 1H), 7.33 (dd, J=2.92,4.43 Hz, 1H), 7.09-7.24 (m, 12H), 3.60-3.79 (m, 4H), 3.37-3.50 (m, 2H),3.34 (d, J=6.03 Hz, 1H), 3.21 (dd, J=5.75, 9.51 Hz, 1H), 2.98 (d,J=10.36 Hz, 1H), 2.67-2.76 (m, 2H), 2.47-2.58 (m, 2H), 2.45 (s, 3H),1.86 (d, J=12.62 Hz, 1H). ¹³C NMR (75 MHz, CDCl₃) δ 173.8, 138.8, 137.8,135.5, 134.7, 134.0, 130.7, 129.6, 129.1, 128.7, 128.6, 128.5, 126.9,126.8, 126.4, 67.0, 58.6, 56.6, 55.9, 50.7, 39.9, 36.4, 35.4, 15.9. MS(ESI) [M]+494.7.

(2S,4S)-1-[(2-Chlorophenyl)methyl]-N—[2-(4-(methylthio)phenyl)ethyl]-4-{[(4-methoxyphenyl)methyl]amino}pyrrolidine-2-carboxamide (46) was prepared according tothe general procedure A as colorless oil (50%). ¹H NMR (300 MHz, CDCl₃)δ 7.47 (br. s., 1H), 7.32-7.38 (m, 1H), 7.15-7.24 (m, 7H), 7.02 (d,J=8.48 Hz, 2H), 6.73 (d, J=8.67 Hz, 2H), 3.71-3.80 (m, 4H), 3.59-3.69(m, 3H), 3.42 (dd, J=6.69, 13.28 Hz, 1H), 3.32-3.38 (m, 1H), 3.26 (d,J=4.14 Hz, 1H), 3.19-3.24 (m, 1H), 2.93 (d, J=9.98 Hz, 1H), 2.61-2.71(m, 2H), 2.51 (dd, J=5.37, 10.08 Hz, 2H), 2.45 (s, 3H), 2.04 (br. s.,2H), 1.78-1.88 (m, 1H). ¹³C NMR (75 MHz, CDCl₃) δ 174.0, 158.2, 137.2,136.5, 135.7, 134.0, 130.8, 130.7, 129.6, 129.5, 128.8, 128.7, 126.9,113.9, 67.2, 59.3, 57.1, 56.1, 55.2, 51.2, 40.0, 37.1, 34.6, 16.0. MS(ESI) [M]⁺524.5.

(2S,4S)-1-[(2-Chlorophenyl)methyl]-N—[2-(4-(methylthio)phenyl)ethyl]-4-{[(4-nitrophenyl)methyl]amino}pyrrolidine-2-carboxamide (47) was prepared according tothe general procedure A as colorless oil (44%). ¹H NMR (300 MHz, CDCl₃)δ 7.93 (d, J=8.67 Hz, 2H), 7.53 (t, J=6.03 Hz, 1H), 7.23-7.28 (m, 1H),7.06-7.18 (m, 8H), 3.51-3.70 (m, 4H), 3.37 (q, J=6.78 Hz, 2H), 3.22 (d,J=3.20 Hz, 1H), 3.17 (dd, J=5.65, 9.80 Hz, 1H), 2.88 (d, J=10.17 Hz,1H), 2.72 (qd, J=7.10, 14.13 Hz, 2H), 2.40-2.49 (m, 2H), 2.39 (s, 3H),1.85 (br. s., 1H), 1.70-1.79 (m, 1H). ¹³C NMR (75 MHz, CDCl₃) δ 173.3,145.7, 145.6, 136.4, 135.4, 134.6, 132.9, 129.4, 128.7, 128.4, 127.8,127.7, 125.9, 125.8, 122.6, 66.1, 58.4, 56.3, 55.3, 50.3, 38.2, 36.1,34.5, 14.9. MS (ESI) [M]⁺539.2.

(2S,4S)-1-[(2-Chlorophenyl)methyl]-N—[2-(3,4-difluorophenyl)ethyl]-4-{[(4-(methylthio)phenyl)methyl]amino}pyrrolidine-2-carboxamide(48) was prepared according to the general procedure A as colorlessliquid (56%). ¹H NMR (300 MHz, CDCl₃) δ 7.58 (br. s., 1H), 7.34 (d,J=7.35 Hz, 1H), 7.15-7.25 (m, 7H), 6.88-7.00 (m, 2H), 6.75-6.83 (m, 1H),3.70-3.80 (m, 1H), 3.59-3.69 (m, 3H), 3.35-3.45 (m, 1H), 3.33 (d, J=6.97Hz, 2H), 3.24 (dd, J=5.65, 9.61 Hz, 1H), 2.96 (d, J=9.80 Hz, 2H),2.60-2.70 (m, 2H), 2.54 (dd, J=5.56, 10.46 Hz, 2H), 2.41-2.49 (m, 4H),1.85 (d, J=13.00 Hz, 1H). ¹³C NMR (75 MHz, CDCl₃) δ 174.1, 137.5, 135.9,135.6, 134.0, 130.6, 129.7, 128.8, 128.8, 126.9, 126.9, 124.5, 124.5,124.4, 124.4, 117.5, 117.3, 117.2, 117.0, 67.1, 59.2, 57.1, 56.1, 51.2,39.6, 36.9, 34.7, 15.9. MS (ESI) [M]⁺530.9.

(2S,4R)-1-[(2-Chlorophenyl)methyl]-4-hydroxypyrrolidine-2-carboxylicacid (49). To a solution of 3 (0.87 g, 3.22 mmol) in methanol (30 ml)was added lithium hydroxide (0.386 g, 16.12 mmol). After stirring atroom temperature for 3 h, the reaction mixture was concentrated invacuo. The residue was dissolved in water and the pH was adjusted to 5with 3N HCl aqueous solution. The reaction mixture was extracted threetimes with ethyl acetate. The combined organic layers were dried overanhydrous magnesium sulfate and concentrated in vacuo to provide theproduct as white solid (0.63 g, 76%). ¹H NMR (300 MHz, CDCl₃) δ7.74-7.79 (m, 2H), 7.37-7.46 (m, 1H), 7.28-7.35 (m, 3H), 7.17-7.23 (m,2H), 5.01 (d, J=5.46 Hz, 1H), 3.74-4.04 (m, 2H), 3.69 (s, 1H), 3.66 (s,1H), 3.29 (dd, J=6.03, 11.11 Hz, 1H), 3.12-3.17 (m, 1H), 2.67-2.73 (m,1H), 2.44 (s, 3H), 2.28 (dd, J=5.46, 7.54 Hz, 2H). MS (ESI) [M]⁺ 256.4,[M−H]⁻ 254.3.

(2S,4R)-1-[(2-Chlorophenyl)methyl]-4-hydroxy-N—[2-(4-nitrophenyl)ethyl]pyrrolidine-2-carboxamide(50). To a solution of 49 (0.825 g, 3.2 mmol) in DMF (10 ml) was added4-nitrophenethylamine (0.981 g, 4.8 mmol), HBTU (1.346 g, 3.5 mmol), anddiisopropylethylamine (2.8 ml, 16.1 mmol). After stirring at roomtemperature for 24 h, the reaction mixture was diluted with ethylacetate, washed water (100 ml each, three times) and brine (100 ml). Theorganic layer was dried over anhydrous magnesium sulfate, andconcentrated in vacuo. The residue was purified by flash columnchromatography (silica gel, methanol/dichloromethane) to provide theproduct as white solid (0.55 g, 42%). ¹H NMR (300 MHz, CDCl₃) δ 8.02 (d,J=8.67 Hz, 2H), 7.53 (t, J=6.03 Hz, 1H), 7.16-7.35 (m, 6H), 4.28-4.37(m, 1H), 3.72-3.87 (m, 2H), 3.56 (t, J=8.10 Hz, 1H), 3.44 (q, J=6.78 Hz,2H), 3.23 (dd, J=4.90, 10.55 Hz, 1H), 2.77-2.89 (m, 2H), 2.53 (dd,J=4.14, 10.55 Hz, 1H), 2.28 (ddd, J=4.24, 8.62, 12.95 Hz, 1H), 1.86-1.98(m, 1H). MS (ESI) [M]⁺404.3.

(3R,5S)-1-[(2-Chlorophenyl)methyl]-5-{[2-(4-nitrophenyl)ethyl]carbamoyl}pyrrolidin-3-yl4-methylbenzene-1-sulfonate (51). To a solution of 50 (0.550 g, 1.36mmol) in dichloromethane (5 ml) was added toluenesulfonyl chloride(0.519 g, 2.72 mmol) and pyridine (5 ml). After stirring at roomtemperature for 24 h, the reaction mixture was concentrated in vacuo.The residue was dissolved in dichloromethane, washed with aqueoussaturated copper sulfate solution twice. The combined organic fractionswere pooled, washed with brine, dried over anhydrous magnesium sulfate,and concentrated in vacuo. The residue was purified by flash columnchromatography (silica gel, ethyl acetate/hexanes) to afford the productas white solid (0.44 g, 58%). ¹H NMR (300 MHz, CDCl₃) δ 7.76 (d, J=8.29Hz, 2H), 7.30-7.37 (m, 4H), 7.15-7.25 (m, 4H), 4.86-4.94 (m, J=5.65 Hz,1H), 3.75 (d, J=2.64 Hz, 2H), 3.39-3.52 (m, 3H), 3.17 (dd, J=4.90, 11.87Hz, 1H), 2.73-2.88 (m, 3H), 2.45 (s, 3H), 2.32-2.42 (m, 1H), 1.90-2.01(m, 1H).

General procedure B: To a solution of 45 (0.12 mmol, 0.068 g) in THF (2ml) was added corresponding amine (1.2 mmol) and triethylamine (0.36mmol, 0.05 ml). The reaction was refluxed for 72 h. After cooling toroom temperature, the solvent was evaporated in vacuo. The residue waspurified by column chromatography (SiO₂, MeOH/DCM) to give the desiredproduct.

(2S,4S)-1-[(2-Chlorophenyl)methyl]-N—[2-(4-nitrophenyl)ethyl]-4-propylamino)pyrrolidine-2-carboxamide(52) was prepared according to the general procedure B as yellow oil(11%). ¹H NMR (300 MHz, CDCl₃) δ 8.02-8.08 (m, 2H), 7.61 (t, J=6.03 Hz,1H), 7.32-7.36 (m, 1H), 7.29 (d, J=8.67 Hz, 2H), 7.16-7.25 (m, 3H),3.60-3.77 (m, 2H), 3.47 (dq, J=3.77, 6.72 Hz, 2H), 3.21-3.29 (m, 2H),2.80-2.95 (m, 3H), 2.38-2.59 (m, 4H), 1.67-1.78 (m, 1H), 1.40 (qd,J=7.29, 14.67 Hz, 3H), 0.87 (t, J=7.35 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃)δ 174.4, 146.8, 146.7, 135.7, 133.9, 130.4, 129.7, 129.4, 128.8, 126.9,123.7, 67.4, 59.8, 57.4, 56.9, 49.9, 39.3, 37.6, 35.6, 23.3, 11.7. MS(ESI) [M]⁺445.6.

(2S,4S)-4-(Butylamino)-1-[(2-chlorophenyl)methyl]-N—[2-(4-nitrophenyl)ethyl]pyrrolidine-2-carboxamide(53) was prepared according to the general procedure B as yellow oil(39%). ¹H NMR (300 MHz, CDCl₃) δ 7.88-7.99 (m, 2H), 7.79 (t, J=5.93 Hz,1H), 7.69 (d, J=7.91 Hz, 1H), 7.29-7.36 (m, 2H), 7.10-7.25 (m, 5H),3.61-3.66 (m, 2H), 3.56 (dd, J=6.69, 14.03 Hz, 1H), 3.44-3.51 (m, 1H),3.34-3.43 (m, 1H), 3.26 (dd, J=6.78, 8.67 Hz, 1H), 3.09 (d, J=10.55 Hz,1H), 2.82 (qd, J=7.08, 13.99 Hz, 2H), 2.61-2.70 (m, 2H), 2.48-2.58 (m,2H), 2.39 (s, 1H), 1.86-1.98 (m, 1H), 1.52 (quin, J=7.49 Hz, 2H), 1.27(qd, J=7.42, 15.05 Hz, 2H), 0.85 (t, J=7.44 Hz, 3H). ¹³C NMR (75 MHz,CDCl₃) δ 173.6, 146.8, 146.5, 142.0, 140.7, 135.1, 133.7, 130.3, 129.6,129.4, 129.0, 128.7, 126.9, 125.7, 123.5, 66.9, 56.7, 55.8, 46.9, 39.3,35.8, 35.2, 30.0, 21.3, 20.1, 13.6. MS (ESI) [M]⁺459.3.

(2S,4S)-1-[(2-Chlorophenyl)methyl]-N—[2-(4-nitrophenyl)ethyl]-4-(pentylamino)pyrrolidine-2-carboxamide (54) was prepared according to the generalprocedure B as colorless oil (59%). ¹H NMR (300 MHz, CDCl₃) δ 8.05 (d,J=8.67 Hz, 2H), 7.62 (t, J=5.93 Hz, 1H), 7.26-7.36 (m, 3H), 7.16-7.25(m, 2H), 3.59-3.76 (m, 2H), 3.47 (td, J=6.57, 12.67 Hz, 2H), 3.21-3.29(m, 2H), 2.81-2.96 (m, 3H), 2.42-2.58 (m, 3H), 1.72 (ddd, J=3.77, 5.75,13.28 Hz, 1H), 1.19-1.44 (m, 6H), 0.87 (t, J=6.88 Hz, 3H). ¹³C NMR (75MHz, CDCl₃) δ 174.4, 146.8, 146.7, 135.7, 133.9, 130.4, 129.7, 129.4,128.8, 126.9, 123.7, 67.4, 59.7, 57.3, 56.9, 48.0, 39.3, 37.6, 35.6,29.9, 29.5, 22.5, 14.0. MS (ESI) [M]⁺473.8.

(2S,4S)-1-[(2-Chlorophenyl)methyl]-4-(hexylamino)-N—[2-(4-nitrophenyl)ethyl]pyrrolidine-2-carboxamide(55) was prepared according to the general procedure B as colorless oil(25%). ¹H NMR (300 MHz, CDCl₃) δ 8.05 (d, J=8.67 Hz, 2H), 7.54 (t,J=5.93 Hz, 1H), 7.27-7.36 (m, 3H), 7.15-7.25 (m, 3H), 3.61-3.77 (m, 2H),3.46 (td, J=6.73, 13.28 Hz, 2H), 3.22-3.33 (m, 2H), 2.75-2.99 (m, 3H),2.43-2.60 (m, 3H), 1.64-1.80 (m, 4H), 1.36-1.45 (m, 2H), 1.21-1.32 (m,6H), 0.84-0.91 (m, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 174.4, 146.7, 135.6,133.9, 130.4, 129.7, 129.4, 128.8, 126.9, 123.7, 67.2, 59.5, 57.2, 57.0,48.0, 39.3, 37.4, 35.5, 31.6, 30.0, 27.0, 22.6, 14.0. MS (ESI)[M]⁺487.4.

(2S,4S)-4-(Benzylamino)-1-[(2-chlorophenyl)methyl]-N—[2-(4-nitrophenyl)ethyl]pyrrolidine-2-carboxamide(56) was prepared according to the general procedure B as colorless oil(81%). ¹H NMR (300 MHz, CDCl₃) δ 7.96-8.03 (m, 2H), 7.62 (t, J=5.75 Hz,1H), 7.15-7.37 (m, 11H), 3.59-3.78 (m, 4H), 3.45 (q, J=6.97 Hz, 2H),3.22-3.34 (m, 2H), 2.96 (d, J=10.17 Hz, 1H), 2.79 (qd, J=7.07, 14.20 Hz,2H), 2.43-2.60 (m, 2H), 1.82 (td, J=3.72, 13.47 Hz, 1H), 1.53 (br. s.,2H). ¹³C NMR (75 MHz, CDCl₃) δ 174.4, 146.7, 146.6, 140.0, 135.7, 134.0,130.5, 129.7, 129.4, 128.8, 128.5, 128.1, 127.2, 126.9, 123.6, 67.2,59.6, 57.4, 56.4, 52.0, 39.2, 37.4, 35.5. MS (ESI) [M]⁺493.8.

(2S,4S)-4-(Benzylamino)-1-[(2-chlorophenyl)methyl]-N—[2-(4-nitrophenyl)ethyl]pyrrolidine-2-carboxamide(57) was prepared according to the general procedure B as colorless oil(13%). ¹H NMR (300 MHz, CDCl₃) δ 8.00 (d, J=8.85 Hz, 1H), 7.16-7.42 (m,12H), 3.62-3.75 (m, 1H), 3.46-3.60 (m, 3H), 3.32 (t, J=8.10 Hz, 1H),3.19-3.28 (m, 1H), 3.14 (dd, J=4.24, 10.64 Hz, 1H), 2.86 (dt, J=4.43,6.83 Hz, 2H), 2.54-2.64 (m, 1H), 2.49 (dd, J=6.03, 13.37 Hz, 1H),2.10-2.17 (m, 2H), 1.88-2.00 (m, 1H). ¹³C NMR (75 MHz, CDCl₃) δ 173.7,146.7, 146.6, 135.5, 133.8, 130.2, 129.7, 129.4, 129.3, 128.8, 128.5,127.7, 127.0, 125.5, 123.7, 67.8, 62.7, 59.5, 57.6, 56.9, 39.3, 38.6,35.4, 34.5. MS (ESI) [M]⁺ 507.2.

(2S,4S)-1-[(2-Chlorophenyl)methyl]-N—[2-(4-nitrophenyl)ethyl]-4-[(2-(phenylethyl)amino]pyrrolidine-2-carboxamide (58) was prepared according to the generalprocedure B as yellow oil (30%). ¹H NMR (300 MHz, CDCl₃) δ 7.99-8.06 (m,1H), 7.48 (t, J=5.93 Hz, 1H), 7.28-7.36 (m, 3H), 7.13-7.25 (m, 7H),3.60-3.71 (m, 2H), 3.48 (td, J=6.90, 13.89 Hz, 1H), 3.28-3.35 (m, 1H),3.18-3.25 (m, 1H), 2.88-3.02 (m, 2H), 2.70-2.83 (m, 6H), 2.44-2.56 (m,2H), 1.85 (d, J=6.97 Hz, 1H), 1.63-1.73 (m, 1H). ¹³C NMR (75 MHz, CDCl₃)δ 174.2, 146.7, 139.7, 135.6, 133.9, 130.3, 129.7, 129.4, 128.8, 128.8,128.6, 128.6, 128.5, 128.5, 126.9, 126.3, 123.6, 67.3, 59.2, 57.2, 56.7,48.9, 39.2, 37.6, 36.2, 35.5. MS (ESI) [M]⁺507.2.

(2S,4S)-1-[(2-Chlorophenyl)methyl]-4-{[(3-methoxyphenyl)methyl]amino}-N—[2-(4-nitrophenyl)ethyl]pyrrolidine-2-carboxamide(59) was prepared according to the general procedure B as yellow oil(20%). ¹H NMR (300 MHz, CDCl₃) δ 8.01 (d, J=8.67 Hz, 2H), 7.60 (t,J=5.93 Hz, 1H), 7.31-7.35 (m, 1H), 7.18-7.25 (m, 6H), 6.74-6.88 (m, 4H),3.79 (s, 3H), 3.69-3.72 (m, 1H), 3.62-3.69 (m, 3H), 3.45 (q, J=6.97 Hz,2H), 3.32 (td, J=2.99, 5.51 Hz, 1H), 3.25 (dd, J=5.37, 9.89 Hz, 1H),2.97 (d, J=9.98 Hz, 1H), 2.80 (q, J=7.35 Hz, 2H), 2.51-2.58 (m, 1H),2.48 (td, J=3.58, 9.98 Hz, 1H), 1.77-1.81 (m, 1H). ¹³C NMR (75 MHz,CDCl₃) δ 174.5, 159.8, 146.7, 146.7, 141.1, 135.6, 134.0, 130.5, 129.7,129.5, 129.4, 128.8, 126.9, 123.6, 120.4, 114.1, 112.4, 67.1, 59.4,57.2, 56.5, 55.2, 51.8, 39.3, 37.2, 35.5. MS (ESI) [M]⁺523.5.

(2S,4S)-1-[(2-Chlorophenyl)methyl]-4-{[(3,4-dimethoxyphenyl)methyl]amino}-N—[2-(4-nitrophenyl)ethyl]pyrrolidine-2-carboxamide(60) was prepared according to the general procedure B as colorless oil(41%). ¹H NMR (300 MHz, CDCl₃) δ 8.00 (d, J=8.67 Hz, 2H), 7.60 (t,J=6.03 Hz, 1H), 7.30-7.36 (m, 1H), 7.16-7.25 (m, 5H), 6.76-6.84 (m, 4H),3.82-3.91 (m, 9H), 3.64-3.72 (m, 2H), 3.58-3.63 (m, 2H), 3.45 (q, J=6.78Hz, 2H), 3.22-3.34 (m, 2H), 2.95 (d, J=10.36 Hz, 1H), 2.79 (q, J=7.10Hz, 2H), 2.45-2.59 (m, 2H), 1.77-1.96 (m, 2H). ¹³C NMR (75 MHz, CDCl₃) δ174.3, 149.1, 148.3, 146.7, 135.7, 134.0, 132.5, 130.5, 129.7, 129.4,128.9, 126.9, 123.6, 120.2, 111.6, 111.3, 111.1, 67.2, 59.6, 57.5, 56.2,55.9, 51.8, 39.2, 37.4, 35.5. MS (ESI) [M]⁺553.7.

(2S,4S)-1-[(2-Chlorophenyl)methyl]-4-{[(4-hydroxyphenyl)methyl]amino}-N—[2-(4-nitrophenyl)ethyl]pyrrolidine-2-carboxamide(61) was prepared according to the general procedure B as colorless oil(27%). ¹H NMR (300 MHz, CDCl₃) δ 7.94 (d, J=8.67 Hz, 2H), 7.69 (t,J=7.72 Hz, 1H), 7.28-7.35 (m, 1H), 7.14-7.25 (m, 4H), 7.05 (d, J=8.48Hz, 2H), 6.61-6.74 (m, 2H), 3.58-3.70 (m, 3H), 3.46 (td, J=6.69, 13.37Hz, 1H), 3.38 (d, J=6.78 Hz, 1H), 3.24 (dd, J=6.12, 9.32 Hz, 1H), 3.01(d, J=10.36 Hz, 1H), 2.74-2.82 (m, 2H), 2.42-2.56 (m, 2H), 1.84 (m, 1H).¹³C NMR (75 MHz, CDCl₃) δ 174.3, 155.7, 146.7, 135.4, 133.8, 130.4,129.8, 129.6, 129.4, 129.0, 128.8, 126.9, 125.8, 123.6, 115.6, 67.2,58.8, 56.9, 56.3, 51.3, 39.3, 36.8, 35.4. MS (ESI) [M]⁺509.4.

(2S,4S)-1-[(2-Chlorophenyl)methyl]-N—[2-(4-nitrophenyl)ethyl]-4-({[4-(trifluoromethyl)phenyl]methyl}amino)pyrrolidine-2-carboxamide (62) was preparedaccording to the general procedure B as colorless oil (9%). ¹H NMR (300MHz, CDCl₃) δ 8.02 (d, J=8.67 Hz, 2H), 7.55 (d, J=8.10 Hz, 3H),7.32-7.38 (m, 3H), 7.16-7.24 (m, 4H), 3.61-3.79 (m, 4H), 3.46 (dd,J=2.54, 6.50 Hz, 2H), 3.26 (dd, J=5.37, 9.89 Hz, 2H), 2.93 (d, J=10.17Hz, 1H), 2.76-2.85 (m, 2H), 2.44-2.59 (m, 2H), 1.84 (dd, J=4.14, 13.38Hz, 1H). ¹³C NMR (75 MHz, CDCl₃) δ 174.2, 146.6, 144.1, 135.5, 134.0,130.5, 129.8, 129.4, 129.0, 128.2, 126.9, 125.3, 123.6, 67.1, 59.6,57.6, 56.5, 51.4, 39.2, 37.3, 35.5. MS (ESI) [M]⁺561.3.

(2S,4S)-1-[(2-Chlorophenyl)methyl]-4-{[(4-chlorophenyl)methyl]amino}-N—[2-(4-nitrophenyl)ethyl]pyrrolidine-2-carboxamide(63) was prepared according to the general procedure B as colorless oil(14%). ¹H NMR (300 MHz, CDCl₃) δ 8.02 (d, J=8.67 Hz, 2H), 7.58 (t,J=6.03 Hz, 1H), 7.14-7.36 (m, 10H), 3.85 (s, 2H), 3.59-3.77 (m, 5H),3.45 (q, J=6.84 Hz, 2H), 3.25 (dd, J=5.37, 9.89 Hz, 2H), 2.92 (d,J=10.17 Hz, 1H), 2.80 (qd, J=6.91, 13.56 Hz, 2H), 2.42-2.57 (m, 2H). ¹³CNMR (75 MHz, CDCl₃) δ 174.3, 146.6, 138.5, 135.6, 134.0, 132.9, 130.5,129.7, 129.6, 129.4, 128.9, 128.6, 128.5, 126.9, 123.7, 67.1, 59.6,57.5, 56.4, 51.3, 39.2, 37.3, 35.5. MS (ESI) [M]⁺527.5.

(2S,4S)-1-[(2-Chlorophenyl)methyl]-4-{[(4-fluorophenyl)methyl]amino}-N—[2-(4-nitrophenyl)ethyl]pyrrolidine-2-carboxamide(64) was prepared according to the general procedure B as colorless oil(55%). ¹H NMR (300 MHz, CDCl₃) δ 8.01 (d, J=8.67 Hz, 2H), 7.59 (t,J=6.12 Hz, 1H), 7.31-7.36 (m, 1H), 7.15-7.28 (m, 7H), 6.95-7.02 (m, 2H),3.57-3.77 (m, 4H), 3.45 (q, J=6.91 Hz, 2H), 3.21-3.32 (m, 2H), 2.93 (d,J=9.98 Hz, 1H), 2.80 (qd, J=6.92, 13.54 Hz, 2H), 2.42-2.58 (m, 2H),1.76-1.86 (m, 1H). ¹³C NMR (75 MHz, CDCl₃) δ 174.3, 146.7, 135.6, 134.0,130.5, 129.7, 129.7, 129.6, 129.4, 128.9, 126.9, 123.6, 115.4, 115.1,67.1, 59.6, 57.5, 56.4, 51.2, 39.2, 37.3, 35.5. MS (ESI) [M]⁺511.3.

1-tert-Butyl 2-methyl (2S,4R)-4-hydroxypyrrolidine-1,2-dicarboxylate(65). To a solution of methyl trans-4-hydroxy-L-proline 5 (27.6 mmol,5.0 g) in 1N NaOH solution (28.5 ml) and 1,4-dioxane (28.5 ml) was addeddropwise Boc₂O (30.3 mmol, 6.6 g) at 0° C. After stirring at roomtemperature for 8 h, the solvent was removed under vacuum. The residuewas dissolved in ethyl acetate, washed with water and brine. The organiclayer was dried over anhydrous MgSO₄, and concentrated in vacuo to givethe desired product as colorless liquid (6.1 g, 90%). ¹H NMR (300 MHz,CDCl₃) δ 4.51 (m, 1H), 4.43 (m, 1H), 3.74 (s, 3H), 3.64 (m, 2H), 2.29(d, J=7.54 Hz, 1H), 2.10 (dd, J=4.52, 8.48 Hz, 1H), 1.44 (m, 9H). MS(ESI) [M+H]⁺ 246.3, [M+Na]⁺268.1.

1-tert-Butyl 2-methyl(2S,4R)-4-[(4-methylbenzenesulfonyl)oxy]pyrrolidine-1,2-dicarboxylate(66). To a solution of 5 (11 g, 45 mmol) in dichloromethane (35 ml) at0° C. was added toluenesulfonyl chloride (10.27 g, 53.9 mmol) andpyridine (35 ml). After stirring under reflux for 24 h, the reactionmixture was concentrated in vacuo and redissolved in dichloromethane. Itwas then washed with a saturated aqueous solution of copper sulfate andbrine. The organic fraction was dried over anhydrous magnesium sulfate,and concentrated in vacuo. The residue was purified by flash columnchromatography (silica gel, ethyl acetate/hexanes) to provide theproduct as light yellow liquid (12.6 g, 70%). ¹H NMR (300 MHz, CDCl₃) δ7.79 (d, J=8.29 Hz, 2H), 7.36 (d, J=8.10 Hz, 2H), 4.99-5.09 (m, 1H),4.32-4.43 (m, 1H), 3.72 (s, 3H), 3.57-3.65 (m, 2H), 2.46 (s, 4H),2.09-2.22 (m, J=8.70 Hz, 1H), 1.36-1.44 (m, 9H). MS (ESI) [M+H]⁺ 400.3,[M+Na]⁺422.3.

1-tert-Butyl 2-methyl (2S,4S)-4-azidopyrrolidine-1,2-dicarboxylate (67).To a solution of 66 (4.28 g, 10.7 mmol) in DMF (40 ml) was added sodiumazide (1.393 g, 21.4 mmol). After heating at 70° C. for 16 h, thereaction mixture was diluted with ethyl acetate, washed with water twiceand brine once.

The organic fraction was dried over anhydrous magnesium sulfate andconcentrated in vacuo to provide the desired product as yellow liquid(2.87 g, 99%). ¹H NMR (300 MHz, CDCl₃) δ 4.30-4.47 (m, 1H), 4.10-4.20(m, 1H), 3.66-3.80 (m, 4H), 3.43-3.54 (m, 1H), 2.47 (ddt, J=6.03, 8.38,13.89 Hz, 1H), 2.13-2.22 (m, 1H), 1.40-1.51 (m, 9H). MS (ESI) [M+H]⁺271.1, [M+Na]⁺293.1.

1-tert-Butyl 2-methyl (2S,4S)-4-aminopyrrolidine-1,2-dicarboxylate (68).To a solution of azide 67 (2.87 g, 10.6 mmol) in THF (46 ml) undernitrogen was added PPh₃ (5.57 g, 21.2 mmol) and water (0.5 ml). Thereaction mixture was refluxed with stirring for 6 h. After the solventwas removed, the residue was dissolved in diethyl ether, treated with0.1 N HCl for 5 min, and then extracted twice with diethyl ether. Theaqueous layer was then treated with 1 N NaOH until pH 10, and thenextracted with dichloromethane. The combined dichloromethane fractionswere dried over anhydrous magnesium sulfate, concentrated in vacuo toafford the desired product as yellow liquid (2.08 g, 80%). ¹H NMR (300MHz, CDCl₃) δ 4.20-4.37 (m, 1H), 3.72-3.79 (m, 3H), 3.63-3.72 (m, 1H),3.50-3.58 (m, 1H), 3.26 (dd, J=4.99, 10.64 Hz, 1H), 2.38-2.53 (m, 1H),1.75-1.86 (m, 1H), 1.39-1.48 (m, 9H). MS (ESI) [M+H]⁺ 245.3.

1-tert-Butyl 2-methyl(2S,4S)-4-{[(4-methoxyphenyl)methyl]amino}pyrrolidine-1,2-dicarboxylate(69). To a solution of 68 (2.08 g, 7.74 mmol) in 1,2-dichloroethane (26ml) was added 4-methoxybenzaldehyde (0.94 ml, 7.74 mmol), sodiumtriacetoxy boron hydride (2.461 g, 11.6 mmol) and acetic acid (0.44 ml,7.74 mmol). After stirring at room temperature for 24 h, the reactionmixture was quenched with saturated sodium bicarbonate solution andextracted three times with dichloromethane. The combined organic layerswere dried over anhydrous magnesium sulfate, and concentrated in vacuo.The residue was purified by flash column chromatography (silica gel,ethyl acetate/hexanes) to provide the product as colorless liquid (2.193g, 73%). ¹H NMR (300 MHz, CDCl₃) δ 7.21 (d, J=8.48 Hz, 2H), 6.85 (d,J=8.67 Hz, 2H), 4.20-4.35 (m, 1H), 3.79 (s, 3H), 3.58-3.75 (m, 6H),3.25-3.37 (m, 2H), 2.29-2.45 (m, 1H), 1.90-2.01 (m, 1H), 1.39-1.48 (m,9H). MS (ESI) [M+H]⁺ 365.9.

1-tert-Butyl 2-methyl(2S,4S)-4-{[(4-methoxyphenyl)methyl][(2,2,2-trichloroethoxy)carbonyl]amino}pyrrolidine-1,2-dicarboxylate(70). To a solution of 69 (0.2 g, 0.5 mmol) in dichloromethane (2.5 ml)at 0° C. was added TrocCl (0.11 ml, 0.77 mmol) and triethylamine (0.14ml, 1.02 mmol). The reaction mixture was brought to room temperature andstirred for 16 h. The reaction mixture was then washed with saturatedaqueous sodium bicarbonate solution. The organic fraction was dried overanhydrous magnesium sulfate and concentrated in vacuo to give theproduct as colorless liquid (0.23 g, quantitative yield). ¹H NMR (300MHz, CDCl₃) δ 7.14 (d, J=8.29 Hz, 2H), 6.85 (d, J=8.48 Hz, 2H),4.73-4.89 (m, 2H), 4.40-4.60 (m, 3H), 4.15-4.26 (m, 1H), 3.79 (s, 3H),3.67-3.75 (m, 4H), 3.37-3.51 (m, 1H), 2.34-2.46 (m, 1H), 2.06-2.23 (m,1H), 1.35-1.47 (m, 9H). MS (ESI) [M+H]⁺ 539.3, [M+Na]⁺563.3.

(2S,4S)-1-[(tert-Butoxy)carbonyl]-4-{[(4-methoxyphenyl)methyl][(2,2,2-trichloroethoxy)carbonyl]amino}pyrrolidine-2-carboxylicacid (71). To a solution of 70 (0.274 g, 0.5 mmol) in methanol (2 ml)was added lithium hydroxide (0.048 g, 2 mmol). After stirring at roomtemperature, methanol was removed under reduced pressure. The residuewas suspended in water and pH was adjusted to 5 by 1N HCl aqueoussolution. The aqueous layer was extracted three times withdichloromethane. The combined organic fractions were dried overanhydrous magnesium sulfate, and concentrated in vacuo to give theproduct as white solid (0.225 g, 84%). ¹H NMR (300 MHz, CDCl₃) δ 9.02(br. s., 1H), 7.14 (d, J=4.33 Hz, 2H), 6.86 (d, J=7.16 Hz, 2H),4.72-4.91 (m, 2H), 4.35-4.63 (m, 3H), 4.16-4.33 (m, 1H), 3.80 (s, 3H),3.70-3.76 (m, 1H), 3.23-3.54 (m, 1H), 2.20-2.52 (m, 2H), 1.41 (d,J=17.52 Hz, 9H). MS (ESI) [M−H]⁻ 523.1.

tert-Butyl-(2S,4S)-4-{[(4-methoxyphenyl)methyl][(2,2,2-trichloroethoxy)carbonyl]amino}-2-[(2-phenylethyl)carbamoyl]pyrrolidine-1-carboxylate(72). To a solution of 71 (2.629 g, 5 mmol) in anhydrous dichloromethane(17 ml) was added phenethylamine hydrochloride (0.7 ml, 5.5 mmol), HBTU(2.086 g, 5.5 mmol), and diisopropylethylamine (3.2 ml, 18 mmol). Afterstirring at room temperature for 16 h, the reaction mixture was washedwith 1N HCl aqueous solution. The organic layer was dried over anhydrousmagnesium sulfate, and concentrated in vacuo. The residue was purifiedby flash column chromatography (silica gel, ethyl acetate/hexanes) toprovide the product as yellow solid (2 g, 64%). ¹H NMR (300 MHz, CDCl₃)δ 7.27 (t, J=3.67 Hz, 2H), 7.12-7.21 (m, 5H), 6.85 (d, J=8.67 Hz, 2H),4.78 (br. s., 2H), 4.33-4.64 (m, 3H), 3.74-3.83 (m, 4H), 3.50 (d, J=5.84Hz, 2H), 2.81-2.88 (m, 2H), 2.17-2.55 (m, 2H), 1.33-1.46 (m, 11H).

2,2,2-Trichloroethyl-N-[(4-methoxyphenyl)methyl]-N-[(3S,5S)-5-[(2-phenylethyl)carbamoyl]pyrrolidin-3-yl]carbamate (73). To a 20% v/v solution oftrifluoroacetic acid in dichloromethane (4.4 ml) was added 72 (2 g, 3.2mmol). The reaction mixture was stirred at room temperature overnight.The reaction mixture was then concentrated in vacuo to provide theproduct as yellow liquid (1.60 g, quantitative yield). MS (ESI)[M]⁺528.7.

General procedure C. To a solution of 73 (0.1 g, 0.19 mmol) in1,2-dichloroethane (4 ml) was added a corresponding aldehyde (0.042 g,0.28 mmol), sodium triacetoxy boron hydride (0.12 g, 0.57 mmol) andacetic acid (1 drop). After stirring at room temperature for 48 h, thereaction mixture was quenched with saturated aqueous sodium bicarbonatesolution, and extracted with three times with dichloromethane. Theorganic extracts were dried over anhydrous magnesium sulfate, andconcentrated in vacuo. The residue was dissolved in methanol (8 ml).Zinc (0.1 g, 1.5 mmol) and acetic acid (2 drops) was added to thereaction mixture. After stirring under reflux for 1 h, the reactionmixture was cooled to room temperature and concentrated in vacuo. Theresidue was purified by flash column chromatography (SiO₂, MeOH/DCM) toafford the desired products.

(2S,4S)-1-Hexyl-4-{[(4-methoxyphenyl)methyl]amino}-N-(2-phenylethyl)pyrrolidine-2-carboxamide(74) was prepared according to the general procedure C as colorless oil(27%). ¹H NMR (300 MHz, CDCl₃) δ 7.50 (t, J=6.03 Hz, 2H), 7.27-7.30 (m,1H), 7.24 (s, 1H), 7.14-7.22 (m, 6H), 6.83-6.87 (m, 2H), 3.79 (s, 3H),3.61 (d, J=3.77 Hz, 2H), 3.41-3.59 (m, 2H), 3.23 (dt, J=3.01, 5.84 Hz,1H), 2.97-3.04 (m, 2H), 2.75-2.82 (m, 2H), 2.33-2.52 (m, 3H), 2.21-2.31(m, 1H), 1.69-1.78 (m, 1H), 1.07-1.38 (m, 9H), 0.89 (t, J=6.78 Hz, 3H).¹³C NMR (75 MHz, CDCl₃) δ 174.8, 158.7, 139.0, 132.3, 129.3, 128.7,128.5, 126.4, 113.8, 67.6, 59.3, 56.2, 56.0, 55.3, 51.3, 39.8, 37.1,35.6, 31.7, 28.8, 27.0, 22.6, 14.0. MS (ESI) [M]⁺438.5.

(2S,4S)-1-(Cyclohexylmethyl)-4-{[(4-methoxyphenyl)methyl]amino}-N-(2-phenylethyl)pyrrolidine-2-carboxamide (75) was prepared according to the generalprocedure C as colorless oil (24%). ¹H NMR (300 MHz, CDCl₃) δ 7.41 (t,J=5.84 Hz, 1H), 7.14-7.25 (m, 6H), 6.83-6.89 (m, 2H), 3.76-3.82 (m, 3H),3.61-3.70 (m, 2H), 3.50-3.59 (m, 2H), 3.24 (td, J=2.76, 5.98 Hz, 1H),2.94-3.01 (m, 2H), 2.80 (qd, J=6.98, 19.00 Hz, 2H), 2.31-2.45 (m, 2H),2.08-2.25 (m, 2H), 1.48-1.81 (m, 8H), 1.23-1.37 (m, 1H), 1.04-1.21 (m,3H), 0.67-0.82 (m, 1H), 0.44 (dd, J=3.20, 11.68 Hz, 1H). ¹³C NMR (75MHz, CDCl₃) δ 173.6, 157.6, 137.7, 131.1, 128.2, 127.6, 127.5, 127.4,125.3, 112.7, 66.9, 61.9, 58.1, 55.2, 54.1, 50.2, 38.5, 35.8, 35.6,34.4, 30.6, 29.6, 25.5, 24.9, 24.8. MS (ESI) [M]⁺450.6.

(2S,4S)-1-Benzyl-4-{[(4-methoxyphenyl)methyl]amino}-N-(2-phenylethyl)pyrrolidine-2-carboxamide(76) was prepared according to the general procedure C as colorless oil(29%). ¹H NMR (300 MHz, CDCl₃) δ 7.44-7.53 (m, 1H), 7.30 (m, 1H),7.12-7.26 (m, 9H), 7.03-7.09 (m, 2H), 6.83 (d, J=8.48 Hz, 2H), 3.73-3.81(m, 4H), 3.46-3.60 (m, 4H), 3.36 (d, J=13.00 Hz, 1H), 3.15-3.26 (m, 2H),2.87 (d, J=10.36 Hz, 1H), 2.72-2.81 (m, 2H), 2.39-2.52 (m, 2H),1.74-1.83 (m, 1H). ¹³C NMR (75 MHz, CDCl₃) δ 174.3, 158.7, 138.9, 138.3,132.2, 129.3, 128.7, 128.6, 128.5, 128.4, 127.2, 126.4, 113.8, 67.1,59.6, 59.2, 56.0, 55.3, 51.3, 39.7, 37.4, 35.6. MS (ESI) [M]⁺444.5.

(2S,4S)-1-[(3-Chlorophenyl)methyl]-4-{[(4-methoxyphenyl)methyl]amino}-N-(2-phenylethyl)pyrrolidine-2-carboxamide(77) was prepared according to the general procedure C as colorless oil(21%). ¹H NMR (300 MHz, CDCl₃) δ 7.45 (t, J=5.18 Hz, 1H), 7.11-7.25 (m,10H), 6.94 (d, J=6.97 Hz, 1H), 6.80-6.86 (m, 2H), 3.78 (s, 3H), 3.73 (d,J=13.37 Hz, 1H), 3.60 (d, J=5.65 Hz, 2H), 3.51-3.57 (m, 1H), 3.41-3.50(m, 1H), 3.30 (d, J=13.56 Hz, 1H), 3.22-3.27 (m, 1H), 3.16 (dd, J=6.12,9.51 Hz, 1H), 2.87 (d, J=10.36 Hz, 1H), 2.78 (dt, J=2.35, 6.92 Hz, 2H),2.36-2.52 (m, 4H), 1.74-1.83 (m, 1H). ¹³C NMR (75 MHz, CDCl₃) δ 173.9,158.9, 140.3, 138.8, 134.3, 131.3, 129.7, 129.5, 128.6, 128.6, 128.5,127.4, 126.6, 126.5, 113.9, 67.3, 58.9, 55.8, 55.3, 51.1, 39.7, 37.2,35.5. MS (ESI) [M]⁺478.3.

(2S,4S)-1-[(4-Chlorophenyl)methyl]-4-{[(4-methoxyphenyl)methyl]amino}-N-(2-phenylethyl)pyrrolidine-2-carboxamide(78) was prepared according to the general procedure C as colorless oil(19%). ¹H NMR (300 MHz, CDCl₃) δ 7.43 (t, J=5.75 Hz, 1H), 7.10-7.26 (m,10H), 6.94 (d, J=8.29 Hz, 2H), 6.81-6.86 (m, 2H), 3.75-3.82 (m, 5H),3.60-3.74 (m, 3H), 3.48-3.60 (m, 4H), 3.26-3.33 (m, 1H), 3.19-3.25 (m,1H), 3.13-3.18 (m, 1H), 2.67-2.90 (m, 4H), 2.35-2.50 (m, 2H), 1.77 (ddd,J=3.49, 5.13, 13.42 Hz, 1H). ¹³C NMR (75 MHz, CDCl₃) δ 174.1, 158.8,138.8, 136.7, 132.9, 132.1, 130.1, 129.8, 129.3, 129.2, 129.1, 128.9,128.8, 128.7, 128.6, 128.6, 128.5, 126.5, 113.9, 113.8, 67.2, 59.1,58.8, 56.0, 55.3, 51.3, 39.6, 37.4, 35.5. MS (ESI) [M]⁺478.5.

(2S,4S)-1-[(2-Methoxyphenyl)methyl]-4-{[(4-methoxyphenyl)methyl]amino}-N-(2-phenylethyl)pyrrolidine-2-carboxamide(79) was prepared according to the general procedure C as colorless oil(25%). ¹H NMR (300 MHz, CDCl₃) δ 7.82 (t, J=5.84 Hz, 1H), 7.28 (d,J=1.70 Hz, 1H), 7.13-7.24 (m, 7H), 7.10 (dd, J=1.51, 7.35 Hz, 1H),6.80-6.92 (m, 4H), 3.87 (d, J=12.81 Hz, 1H), 3.77 (s, 3H), 3.72 (s, 3H),3.51-3.65 (m, 3H), 3.32-3.44 (m, 2H), 3.15-3.24 (m, 2H), 2.85 (d,J=10.17 Hz, 1H), 2.77 (dt, J=1.98, 7.21 Hz, 2H), 2.42-2.54 (m, 2H), 1.82(ddd, J=3.30, 5.75, 13.56 Hz, 1H). ¹³C NMR (75 MHz, CDCl₃) δ 173.4,157.7, 156.6, 138.1, 131.2, 129.6, 128.3, 127.7, 127.6, 127.5, 125.4,125.3, 119.5, 112.8, 109.5, 65.8, 58.2, 54.8, 54.3, 54.2, 53.6, 50.2,39.2, 36.4, 34.9. MS (ESI) [M]⁺474.6.

(2S,4S)-1-[(3-Methoxyphenyl)methyl]-4-{[(4-methoxyphenyl)methyl]amino}-N-(2-phenylethyl)pyrrolidine-2-carboxamide(80) was prepared according to the general procedure C as colorless oil(45%). ¹H NMR (300 MHz, CDCl₃) δ 7.54 (br. s., 1H), 7.31 (d, J=8.48 Hz,2H), 7.10-7.24 (m, 6H), 6.69-6.87 (m, 4H), 3.80-3.90 (m, 2H), 3.75 (d,J=15.82 Hz, 0H), 3.01-3.65 (m, 6H), 2.74-2.87 (m, 2H), 2.34-2.52 (m,2H), 1.87-2.04 (m, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 171.3, 157.7, 157.5,136.5, 128.8, 127.3, 126.6, 126.3, 124.2, 122.9, 122.6, 118.9, 112.2,110.9, 107.3, 64.1, 56.0, 54.2, 53.1, 53.1, 52.7, 47.2, 38.1, 33.1,32.5. MS (ESI) [M]⁺474.9.

(2S,4S)-1-[(3,4-Dimethoxyphenyl)methyl]-4-{[(4-methoxyphenyl)methyl]amino}-N-(2-phenylethyl)pyrrolidine-2-carboxamide(81) was prepared according to the general procedure C as colorless oil(55%). ¹H NMR (300 MHz, CDCl₃) δ 7.02-7.26 (m, 3H), 6.80-6.94 (m, 6H),6.46-6.79 (m, 2H), 3.83-3.91 (m, 12H), 3.75-3.79 (m, 2H), 3.43-3.73 (m,2H), 2.95-3.33 (m, 1H), 2.68-2.91 (m, 2H), 1.68-2.50 (m, 4H). ¹³C NMR(75 MHz, CDCl₃) δ 172.1, 146.8, 146.2, 131.4, 127.0, 126.8, 126.4,126.3, 126.2, 126.1, 124.0, 118.4, 117.0, 111.5, 111.5, 108.8, 108.2,62.7, 56.6, 53.6, 53.5, 52.9, 49.6, 48.9, 37.6, 35.0, 33.2, 30.7. MS(ESI) [M]⁺504.7.

(2S,4S)-4-{[(4-Methoxyphenyl)methyl]amino}-1-[(2-methylphenyl)methyl]-N-(2-phenylethyl)pyrrolidine-2-carboxamide(82) was prepared according to the general procedure C as colorless oil(19%). ¹H NMR (300 MHz, CDCl₃) δ 7.36 (t, J=5.75 Hz, 1H), 7.12-7.19 (m,7H), 7.07-7.11 (m, 2H), 6.84 (d, J=8.48 Hz, 2H), 3.75-3.80 (m, 4H),3.51-3.70 (m, 5H), 3.45-3.50 (m, 1H), 3.29-3.38 (m, 1H), 3.17-3.28 (m,2H), 2.95 (d, J=9.98 Hz, 1H), 2.67 (dt, J=2.73, 7.02 Hz, 2H), 2.42-2.51(m, 2H), 2.21-2.27 (m, 3H), 2.19 (d, J=3.01 Hz, 1H), 1.74-1.82 (m, 1H).¹³C NMR (75 MHz, CDCl₃) δ 172.2, 156.5, 136.6, 134.3, 134.0, 129.9,128.0, 127.1, 126.9, 126.5, 126.4, 126.3, 124.9, 124.1, 123.8, 111.6,111.6, 65.6, 57.4, 55.4, 53.9, 53.0, 49.1, 37.5, 35.2, 33.3, 16.9. MS(ESI) [M]⁺458.4.

(2S,4S)-4-{[(4-Methoxyphenyl)methyl]amino}-1-[(4-methylphenyl)methyl]-N-(2-phenylethyl)pyrrolidine-2-carboxamide(83) was prepared according to the general procedure C as colorless oil(26%). ¹H NMR (300 MHz, CDCl₃) δ 7.48 (t, J=5.84 Hz, 1H), 7.13-7.25 (m,6H), 7.07 (d, J=7.91 Hz, 2H), 6.92-6.98 (m, 2H), 6.81-6.86 (m, 2H), 3.78(s, 3H), 3.72 (d, J=13.00 Hz, 1H), 3.57 (d, J=3.77 Hz, 2H), 3.46-3.54(m, 2H), 3.33 (d, J=13.00 Hz, 1H), 3.22 (d, J=3.20 Hz, 1H), 3.17 (dd,J=6.03, 9.61 Hz, 1H), 2.87 (d, J=10.17 Hz, 1H), 2.76 (dt, J=4.43, 6.92Hz, 2H), 2.39-2.50 (m, 2H), 2.33 (s, 3H), 1.73-1.82 (m, 1H). ¹³C NMR (75MHz, CDCl₃) δ 173.1, 157.5, 137.7, 135.6, 133.9, 130.8, 128.1, 127.8,127.4, 127.3, 127.2, 125.2, 112.6, 65.8, 58.0, 57.8, 54.8, 54.0, 50.0,38.5, 36.1, 34.4, 19.8. MS (ESI) [M]⁺458.4.

(2S,4S)-1-[(4-Hydroxyphenyl)methyl]-4-{[(4-methoxyphenyl)methyl]amino}-N-(2-phenylethyl)pyrrolidine-2-carboxamide(84) was prepared according to the general procedure C as colorless oil(9%). ¹H NMR (300 MHz, CDCl₃) δ 7.50 (t, J=5.56 Hz, 1H), 7.12-7.23 (m,7H), 6.80-6.89 (m, 4H), 6.60 (d, J=8.29 Hz, 2H), 3.74 (s, 3H), 3.61-3.70(m, 3H), 3.50 (td, J=7.18, 14.46 Hz, 2H), 3.31 (s, 1H), 3.21 (d, J=13.00Hz, 1H), 3.09 (dd, J=6.40, 9.42 Hz, 1H), 2.92 (d, J=10.36 Hz, 1H),2.69-2.85 (m, 3H), 2.36-2.48 (m, 2H), 1.83 (dd, J=4.24, 13.47 Hz, 1H).¹³C NMR (75 MHz, CDCl₃) δ 174.1, 159.5, 155.7, 138.7, 130.2, 129.9,129.0, 128.7, 128.6, 128.6, 126.5, 115.4, 114.2, 66.5, 58.2, 57.8, 55.4,55.2, 50.3, 40.0, 35.6, 35.4. MS (ESI) [M]⁺460.5.

(2S,4S)-1-{[4-(Dimethylamino)phenyl]methyl}-4-{[(4-methoxyphenyl)methyl]amino}-N-(2-phenylethyl)pyrrolidine-2-carboxamide(85) was prepared according to the general procedure C as colorless oil(3%). ¹H NMR (300 MHz, CDCl₃) δ 7.37 (br. s., 1H), 7.28 (d, J=1.88 Hz,0H), 7.15-7.22 (m, 4H), 6.95 (d, J=8.67 Hz, 1H), 6.82-6.86 (m, 1H), 6.63(d, J=8.67 Hz, 1H), 3.78 (s, 2H), 3.61-3.69 (m, 2H), 3.44-3.52 (m, 1H),3.32 (d, J=12.81 Hz, 1H), 3.25 (d, J=5.09 Hz, 1H), 3.15 (dd, J=5.84,9.61 Hz, 1H), 2.89-2.97 (m, 5H), 2.76 (td, J=3.49, 6.97 Hz, 1H),2.46-2.53 (m, 1H), 2.36-2.45 (m, 1H), 1.81 (s, 1H). ¹³C NMR (75 MHz,CDCl₃) δ 172.6, 148.1, 137.1, 127.8, 127.7, 126.9, 126.7, 124.6, 124.0,112.1, 110.7, 64.7, 56.9, 54.1, 53.4, 49.1, 38.8, 38.1, 35.0, 33.8,29.0. MS (ESI) [M]⁺487.6.

(2S,4S)-1-[(4-Cyanophenyl)methyl]-4-{[(4-methoxyphenyl)methyl]amino}-N-(2-phenylethyl)pyrrolidine-2-carboxamide(86) was prepared according to the general procedure C as colorless oil(27%). ¹H NMR (300 MHz, CDCl₃) δ 7.52 (d, J=8.29 Hz, 2H), 7.44 (t,J=5.75 Hz, 1H), 7.08-7.25 (m, 9H), 6.84 (d, J=8.67 Hz, 2H), 3.75-3.82(m, 4H), 3.50-3.65 (m, 4H), 3.38 (d, J=13.75 Hz, 1H), 3.23-3.29 (m, 1H),3.20 (dd, J=5.93, 9.70 Hz, 1H), 2.70-2.89 (m, 3H), 2.42-2.51 (m, 1H),2.37 (dd, J=5.65, 9.98 Hz, 1H), 1.73-1.83 (m, 1H). ¹³C NMR (75 MHz,CDCl₃) δ 228.8, 173.8, 158.8, 143.8, 138.8, 132.2, 129.3, 128.9, 128.7,128.6, 126.5, 113.9, 67.5, 59.2, 59.1, 56.1, 55.3, 51.4, 39.5, 37.4,35.4. MS (ESI) [M]⁺469.4.

(2S,4S)-4-{[(4-Methoxyphenyl)methyl]amino}-1-(naphthalen-2-ylmethyl)-N-(2-phenylethyl)pyrrolidine-2-carboxamide(87) was prepared according to the general procedure C as colorless oil(23%). ¹H NMR (300 MHz, CDCl₃) δ 7.80-7.84 (m, 1H), 7.72-7.79 (m, 2H),7.58 (s, 1H), 7.43-7.55 (m, 3H), 7.10-7.24 (m, 8H), 6.82 (d, J=8.67 Hz,2H), 3.93 (d, J=13.00 Hz, 1H), 3.77 (s, 3H), 3.43-3.59 (m, 5H),3.21-3.29 (m, 2H), 2.89 (d, J=10.17 Hz, 1H), 2.76 (dt, J=3.20, 6.88 Hz,2H), 2.42-2.55 (m, 2H), 1.80 (ddd, J=3.49, 5.65, 13.47 Hz, 1H). ¹³C NMR(75 MHz, CDCl₃) δ 172.5, 157.1, 137.2, 134.1, 131.7, 131.1, 130.1,127.7, 127.0, 126.9, 126.5, 126.1, 126.0, 125.4, 125.0, 124.8, 124.5,124.1, 112.2, 65.6, 58.1, 57.4, 54.2, 53.6, 49.5, 38.1, 35.7, 33.9. MS(ESI) [M]⁺494.4.

(2S,4S)-1-(2H-1,3-Benzodioxol-5-ylmethyl)-4-{[(4-methoxyphenyl)methyl]amino}-N-(2-phenylethyl)pyrrolidine-2-carboxamide(88) was prepared according to the general procedure Cas colorless oil(11%). ¹H NMR (300 MHz, CDCl₃) δ 7.35 (br. s., 1H), 7.14-7.25 (m, 7H),6.84 (d, J=8.67 Hz, 2H), 6.69 (d, J=7.91 Hz, 1H), 6.59 (s, 1H), 6.53 (d,J=7.72 Hz, 1H), 5.92 (d, J=2.07 Hz, 2H), 3.78 (s, 3H), 3.61-3.70 (m,3H), 3.51 (td, J=6.64, 13.09 Hz, 2H), 3.27 (d, J=13.00 Hz, 2H), 3.13(dd, J=5.84, 9.61 Hz, 1H), 2.92 (d, J=10.93 Hz, 1H), 2.79 (dt, J=3.77,6.97 Hz, 2H), 2.37-2.49 (m, 2H), 1.82 (d, J=16.39 Hz, 2H). ¹³C NMR (75MHz, CDCl₃) δ 172.7, 146.3, 145.4, 137.4, 130.5, 128.3, 127.3, 127.2,125.1, 120.3, 112.6, 107.6, 106.7, 99.5, 97.0, 65.4, 57.6, 57.1, 54.4,53.9, 49.4, 38.4, 35.2, 34.1. MS (ESI) [M]⁺488.6.

Calcium mobilization assay. Two individual stable cell lines werecreated by over-expressing human NPFFR1 and NPFFR2 receptors inCHO-RD-HGA16 (Molecular Devices) cells. The day before the assay, cellswere plated into 96-well black-walled assay plates at 30,000 cells/well(100 μL volume) in Ham's F12 supplemented with 10% fetal bovine serum,100 units of penicillin/streptomycin, and 100 g/mL Normocin™.

The cells were incubated overnight at 37° C., 5% CO₂. Prior to theassay, Calcium 5 dye (Molecular Devices) was reconstituted according tothe manufacturer instructions. The reconstituted dye was diluted 1:40 inwarm assay buffer (1×HBSS, 20 mM HEPES, 2.5 mM probenecid, pH 7.4 at 37°C.). Growth medium was removed and the cells were gently washed with 100μL of warm assay buffer. The cells were incubated for 45 minutes at 37°C., 5% CO₂ in 200 μL of the diluted Calcium 5 dye. A singleconcentration of each test compound was prepared at 10× the desiredfinal concentration in 2.25% BSA/8% DMSO/assay buffer. Serial dilutionsof NPFF were prepared at 10× the desired final concentration in 0.25%BSA/1% DMSO/assay buffer, aliquoted into 96-well polypropylene plates,and warmed to 37° C. After the dye-loading incubation period, the cellswere pre-treated with 25 μL of the test compounds and incubated for 15min at 37° C. After the pre-treatment incubation period, the plate wasread with a FlexStation® II (Molecular Devices). Calcium-mediatedchanges in fluorescence were monitored every 1.52 seconds over a 60second time period, with the FlexStation® II adding 25 μL of the NPFFserial dilutions at the 19 second time point (excitation at 485 nm,detection at 525 nm). Peak kinetic reduction (SoftMax, MolecularDevices) relative fluorescent units (RFU) were plotted against the logof compound concentration.

Data were fit to a three-parameter logistic curve to generate EC₅₀values (GraphPad Prism, GraphPad Software, Inc., San Diego, CA).Apparent K_(e) values were calculated using the equationK_(e)=[L]/((EC₅₀ ⁺/EC₅₀ ⁻)−1) where [L] is the concentration of testcompound, EC₅₀ ⁺ is the EC₅₀ of NPFF with test compound, and EC₅₀ ⁻ isthe EC₅₀ of NPFF alone. K_(e) values were considered valid when the EC₅₀⁺/EC₅₀ ⁻ ratio was at least 4.

384-Well High Throughput Screening

Stable human NPFFR1 CHO-RD-HGA16 cells were plated in 30 μL/well volumeat 5,000 cells/well in Ham's F12 medium supplemented with 1% FBS and 100units penicillin/streptomycin in 384-well Greiner & Clear® black walledmicroplates using a MicroFlo™ Select dispenser fitted with a 5 μLcassette (BioTek). The plated cells were incubated overnight at 37° C.,5% C₀2, 95% relative humidity. The next day, compound test plates wereprepared by diluting previously replicated library daughter plates withassay buffer to achieve a 100 μM (10× desired final concentration)working solution and filling columns 1, 2, 23, and 24 with positive andnegative controls. An additional compound test plate containing the NPFFEC₆₀ concentration (250 nM prepared at 10× the desired finalconcentration in 1% DMSO/assay buffer) was prepared for the antagonistportion of the screen. Calcium 5 dye (Bulk Kit, Molecular Devices),reconstituted according to the manufacturer's instructions, was diluted1:20 in pre-warmed (37° C.) assay buffer (1×HBSS, 20 mM HEPES, 2.5 mMprobenecid, pH 7.4 at 37° C.) and 30 μL was added to the plate with theBiomek NX, which was then incubated for 45 minutes at 37° C., 5% CO₂,95% relative humidity. Using the Biomek NX, the dye loaded plate waspretreated with 8.5 μL of 8% DMSO/assay buffer and incubated for 15minutes at 37° C., 5% CO₂, 95% relative humidity. After this incubationperiod, the plate was read with the Tetra to evaluate agonist activity.Calcium-mediated changes in fluorescence were monitored every 1 secondover a 60 second time period, with the Tetra adding 8.5 μL from thecompound plate at the 10 second time point (excitation at 470-495 nm,detection at 515-575 nm). The cell plate was then incubated for another15 minutes at 37° C., 5% CO₂, 95% relative humidity after which it wasread with the Tetra to evaluate antagonist activity. Calcium-mediatedchanges in fluorescence were monitored every 1 second over a 60 secondtime period, with the Tetra adding 8.5 μL from the NPFF EC₆₀ plate atthe 10 second time point (excitation at 470-495 nm, detection at 515-575nm). Data was exported from ScreenWorks (Molecular Devices) using theresponse over baseline (ROB) statistic which presents data as afold-response compared with the baseline sample. Percent inhibition wascalculated using the equation (1−(cmpd ROB/NPFF EC₆₀ ROB))×100.

cAMP assay. Stable human NPFFR1-CHO (ES-491-C) and NPFFR2-CHO (ES-490-C)cell lines were purchased from PerkinElmer and used with the Lance®Ultra kit (TRF0262) to detect cAMP accumulation in low volume 96-wellplates. Stimulation buffer containing 1×HBSS, 5 mM HEPES, 0.1% BSAstabilizer, and 0.5 mM IBMX was prepared and titrated to 7.4 at roomtemperature. Serial dilutions of the agonist control NPFF were preparedat 8× the desired final concentration in 2% DMSO/stimulation buffer and2.5 μL was added to the assay plate.

A single concentration of each test compound was prepared at 4× thedesired final concentration in 2% DMSO/stimulation buffer and 5 μL wasadded to the assay plate. The EC₈₀ concentration of forskolin (1 μM) wasprepared at 8× in 2% DMSO/stimulation buffer and 2.5 μL was added to theassay plate.

Cells were lifted with versene and spun at 270 g for 10 minutes. Thecell pellet was resuspended in stimulation buffer and 4,000 cells (10μL) were added to each well. After incubating for 30 min at RT, Eu-cAMPtracer and uLIGHT-anti-cAMP working solutions were added per themanufacturer's instructions. After incubation at RT for 1 hour, theTR-FRET signal (ex 337 nm) was read on a CLARIOstar multimode platereader (BMG Biotech, Cary NC).

Fluorescence values at 665 nm were plotted against the log of compoundconcentration using a nonlinear regression analysis to generate EC₅₀values (GraphPad Prism, GraphPad Software, Inc., San Diego CA). K_(e)values were calculated using the same equation as described in thecalcium mobilization methods.

Radioligand binding assay. Binding assays were performed according toPerkinElmer's protocol in a final volume of 500 μL of assay buffer (50mM Tris-HCl, 1 mM MgCl₂, 60 mM NaCl, 0.5% BSA, pH 7.4). For NPFF1, theassay mixture contained 25 μL of 0.065 nM [¹²⁵I]NPFF (PerkinElmer,K_(D)=0.11 nM, NEX381), 25 μL of test compounds (prepared at 8× thefinal desired concentration in 8% DMSO/assay buffer), and 150 μL ofCHO-hNPFFR1 membranes (1 μg protein/well, PerkinElmer, RBHNF1M400UA).Specific binding was defined as the difference between [¹²⁵I]NPFFbinding in the absence and presence of 100 nM final nonradiolabeledNPFF. After incubating at 27° C. for 120 minutes, the binding assay wasterminated by vacuum filtration onto Unifilter GF/C glass-fiber filters(pre-soaked in 0.1% PEI) using a Brandel (Gaithersburg, MD, USA) 96-wellharvester, followed by three washes with ice-cold wash buffer (50 mMTris-HCl, 0.1% BSA, pH 7.4). The filter plate was dried for 1 hr at 55°C. Microscint 20 (50 μL) was added to each well and filter-boundradioactivity was counted on a Packard TopCount NXT microplatescintillation and luminescence counter. Percentage of specific[¹²⁵I]NPFF binding was plotted against the log of compoundconcentration.

Data were fit to a one site (fit Ki) competitive binding model, logEC50=log(10{circumflex over ( )}log Ki*(1+RadioligandNM/HotKdNM)), togenerate Ki values for the test compounds using GraphPad Prism (GraphPadSoftware, Inc., San Diego CA). NPFF2 binding assays were conducted withthe same protocol, except that 0.1 nM [¹²⁵I]NPFF (PerkinElmer,K_(D)=0.15 nM, NEX381) and CHO-hNPFFR2 membranes (1 μg protein/well,PerkinElmer, RBHNF2M400UA) were used.

Kinetic solubility assay. A 10 μL of test compound stock solution (10 mMDMSO) was combined with 490 μL of PBS (potassium phosphate monobasic 1mM, sodium phosphate dibasic 3 mM and sodium chloride 155 mM buffer).The solution was agitated on a VX-2500 multi-tube vortexer (VWR) for 2hours at room temperature. Following agitation, the sample was filtratedon a glass-fiber filter (1 μm) and the eluate was diluted 200-fold witha mixture of acetonitrile: water (1:1). On each experimental occasion,nicardipine and imipramine were assessed as reference compounds for lowand high solubility, respectively. All samples were assessed intriplicate and analyzed by LC-MS/MS using electrospray ionizationagainst standards prepared in the same matrix.

Bidirectional MDCK-MDR1 permeability assay. MDCK-mdr1 cells at passage 5were seeded onto permeable polycarbonate supports in 12-well Costar®Transwell® plates and allowed to grow and differentiate for 3 days. Onday 3, culture medium (DMEM supplemented with 10% FBS) was removed fromboth sides of the transwell inserts and cells were rinsed with warmHBSS. After the rinse step, the chambers were filled with warm transportbuffer (HBSS containing 10 mM HEPES, 0.25% BSA, pH 7.4) and the plateswere incubated at 37° C. for 30 min prior to TEER (Trans EpithelialElectric Resistance) measurements.

The buffer in the donor chamber (apical side for A-to-B assay,basolateral side for B-to-A assay) was removed and replaced with theworking solution (10 μM test article in transport buffer). The plateswere then placed at 37° C. under light agitation. At designated timepoints (30, 60 and 90 min), an aliquot of transport buffer from thereceiver chamber was removed and replenished with fresh transportbuffer. Samples were quenched with ice-cold ACN containing internalstandard and then centrifuged to pellet protein. Resulting supernatantsare further diluted with 50/50 ACN/H₂O (H₂O only for Atenolol) andsubmitted for LC-MS/MS analysis. Reported apparent permeability (Papp)values were calculated from single determination. Atenolol andpropranolol were tested as low and moderate permeability references.Bidirectional transport of digoxin was assessed to demonstrate Pgpactivity/expression.

The apparent permeability (Papp, measured in cm/s) of a compound isdetermined according to the following formula:

${Papp} = \frac{({dQ})/({dt})}{A*C*60}$

-   -   dQ/dt is the net rate of appearance in the receiver compartment    -   A is the area of the Transwell measured in cm² (1.12 cm²)    -   Ci is the initial concentration of compound added to the donor        chamber    -   60 is the conversion factor for minutes to seconds.

In Vivo Pharmacology.

Animals. Adult male (n=18) Sprague-Dawley rats (Harlan, Indianapolis,IN) weighing 225-300 g were individually housed on a 12/12-hourlight/dark cycle with behavioral experiments conducted during the lightperiod. Rats had free access to food and water except during testsessions, and were maintained and experiments were conducted inaccordance with guidelines of the International Association for theStudy of Pain (Zimmermann, M., Ethical guidelines for investigations ofexperimental pain in conscious animals, Pain, 16, 109-110, 1983) andwith the 2011 Guide for the Care and Use of Laboratory Animals(Institute of Laboratory Animal Resources on Life Sciences, NationalResearch Council, National Academy of Sciences, Washington, DC), andwere approved by the Institutional Animal Care and Use Committee,University at Buffalo, the State University of New York (Buffalo, NY).

Drugs. Fentanyl was purchased from Sigma-Aldrich (St. Louis, MO),dissolved in 0.9% saline, and injected subcutaneously in a volume of 1ml/kg. Compounds 16 and 33 were dissolved in a vehicle of 20% dimethylsulfoxide in saline and injected intraperitoneally in a volume of 1ml/kg.

Fentanyl-induced hyperalgesia. Nociceptive thresholds were measuredusing calibrated von Frey filaments (1.4-26 g; North Coast Medical,Morgan Hill, CA). Rats (n=6 per group) were placed in elevated plasticchambers with a wire mesh floor (IITC Life Science Inc., Woodland Hills,CA) and allowed to habituate prior to testing. Filaments were appliedperpendicularly to the medial plantar surface of the hind paw from belowthe mesh floor in an ascending order of filament force, beginning withthe lowest filament. Filaments were applied until buckling occurred forapproximately two seconds. Mechanical paw withdrawal thresholds (PWTs)correspond to the lowest force that elicited a withdrawal of the hindpaw in at least two out of three applications. Forces larger than 26 gwould physically elevate the non-CFA-treated paw and did not reflectpain-like behavior.

After a nociceptive baseline was established for each rat on day priorto and the day of fentanyl treatment (D⁻¹ and D₀), four subcutaneousinjections of 0.06 mg/kg fentanyl each were injected at 15 min intervalsfor a total dose of 0.24 mg/kg. In one group of rats, PWT measurementswere taken on days 1-5 to monitor the onset of and recovery from thefentanyl-induced hyperalgesia. In the other two groups of rats,antinociceptive dose-effect curves for compounds 16 and 33 wereestablished on day 1 using a multi-cycle cumulative dosing procedure, inwhich measurements were taken immediately prior to drug administrationthen 30 min after drug administration before the next injection, andcontinued for doses ranging from 3.2-32 mg/kg.

Data analysis. PWTs were averaged within each group and plotted as afunction of dose. Repeated measures one-way ANOVAs, with time ortreatment entered as the within-subject factor, followed by Bonferroni'spost-hoc test were used to determine the statistical significances.P<0.05 was considered statistically significant for all tests.

Pharmacological Evaluation of Compounds 16 and 33

Compounds 16 and 33 were identified as potent NPFF1 antagonists, andselected for further characterization and assessment.

The concentration-response curve of compound 16 in the NPFF1 and NPFF2calcium mobilization assays is shown in FIG. 4 , in which graph A showsthe antagonist activity of compound 16 in the NPFF1 calcium mobilizationfunctional K_(e) assay, and graph B shows the antagonist activity ofcompound 16 in the NPFF2 calcium mobilization functional K_(e) assay.Graph A shows the concentration-response curves of NPFF alone (∘) andNPFF+5 μM final 16 (□) in stable NPFF1-RD-HGA16 cells. Graph B shows theconcentration-response curves of NPFF alone (∘) and NPFF+10 μM final 16(□) in stable NPFF2-RD-HGA16 cells. The right shift of the NPFF curve inthe presence of the test compound was used to calculate K_(e) values.Representative data from one experiment are shown and each data point ismean±SD of duplicate determinations.

The concentration-response curves of compounds 16 and 33 in the NPFF1and NPFF2 cAMP assays are shown in FIG. 5 , in which graph A shows theantagonist activity of compounds 16 and 33 in the NPFF1 cAMP functionalK_(e) assay, and graph B shows the antagonist activity of compounds 16and 33 in the NPFF2 cAMP functional K_(e) assay. Graph A shows theconcentration-response curves of NPFF alone (∘), NPFF+4 μM final 16 (□),and NPFF+2 μM final 33 (⋄) in stable NPFF1-CHO cells. Graph B shows theconcentration-response curves of NPFF alone (∘), NPFF+10 μM final 16(□), and NPFF+10 μM final 33 (⋄) in stable NPFF2-CHO cells. The rightshift of the NPFF curve in the presence of the test compound was used tocalculate K_(e) values. Each data point is mean±SEM of at least N=3conducted in duplicate.

In the cAMP assay, the NPFF+16 curve had a Hill slope of 1.3 in bothNPFF1 and NPFF2 cells while the NPFF+33 curve had a Hill slope of 1.4and 1 in NPFF1 and NPFF2 cells, respectively, thus indicating that theactivity of these compounds is not due to the formation of aggregates.These results correlate with those from the calcium mobilization assayswhere the NPFF+16 curve had a Hill slope of 1.7 and 1.1 in NPFF1 and FF2cells, respectively, and the NPFF+33 curve had a Hill slope of 1.4 inboth cell lines.

Table 6 below sets out selected physicochemical and preliminary ADME(absorption, distribution, metabolism, and excretion) propertiesdetermined for compounds 1, 16, and 33.

TABLE 6 Desired Parameter value Compound 1 Compount 16 Compound 33Molecular weight <500 418 444 523 ClogP 1-4 3.57 4.59 4.37 PSA <70 69.753.6 54.4 PKa <8 8.6, 5.0 9.1, 4.8 9.1, 4.8 HBD <3 2 2 2 HBA <7 4 5 8Solubility (μM) >60 N.D. 146.8 ±6.9 45.9 ±7.7 Papp (10⁻⁶ cm/sec) >2 N.D.7.6 2.7 A-to-B Papp (10⁻⁶ cm/sec) N.D. 6.7 3.6 B-to-A Efflux ratio <2.5N.D. 0.9 1.3 HBD: H-bond donor; HBA: H-bond acceptor. N.D.: Notdetermined.

The compounds 16 and 33 were tested as free base forms in kineticsolubility and bidirectional MDCK-MDR1 permeability assays. As seen inTable 6, compound 16 was soluble in aqueous solutions, displaying akinetic solubility of 146.8±6.9 μM (Mean±% CV) which falls in the rangeof compounds with good solubility, while compound 33 had a lowersolubility of 45.9±7.7 μM, which is expected for a larger molecule witha higher molecular weight. Moreover, these compounds contain multipleprotonable nitrogen atoms which can be converted to salt forms, toenhance solubility and bioavailability.

One of the major challenges for central nervous system (CNS) drugs istheir ability to cross the blood-brain barrier (BBB) and reach the CNS.For the majority of drugs, the BBB permeability is affected by twofactors: the ability to permeate through the BBB passively and theavoidance of being effluxed out by the transport proteins such asP-glycoprotein. Compounds 16 and 33 were evaluated in the bidirectionaltransport assay using MDCK-MDR1 cells which are stably transfected withhuman MDR1 cDNA and express a higher level of the P-glycoprotein (Pgp)compared to the wild type. Table 6 shows that compound 16 traversed thecell barrier from the apical (A) to basolateral (B) at a rate of7.6×10⁻⁶ cm/s, and the reverse direction B to A at a rate of 6.7×10⁻⁶cm/s, demonstrating a moderate BBB permeability (within the range of3−6×10⁻⁶ cm/s). Compound 33 is also able to penetrate the BBB albeit atlower permeability compared to compound 16. Both compounds were not Pgpsubstrates as indicated by the efflux ratio (P_(B→A)/P_(A→B)). Together,the data evidence the character of these compounds as CNS positiveligands.

Anti-Hyperalgesia Effects of Compounds 16 and 33 in Rats

Compounds 16 and 33 were tested in a fentanyl-induced hyperalgesia modelin rats, with the results shown in FIG. 6 , in which graph A showsresults for fentanyl-induced mechanical hyperalgesia, and graph B showsthe anti-hyperalgesic effects of compounds 16 and 33 (N=6 per group).The ordinate in the graphs is paw withdrawal threshold, in grams, andthe abscissa is time. P<0.05 compared to pre-fentanyl treatment (Day 0)or compared to “V” (vehicle) treatment.

Prior to fentanyl treatment, administered i.p., the test rats displayeda mean paw withdrawal threshold (PWT) of 24.2±1.8 g, which decreased to5.7±0.8 g on day 1 after fentanyl administration. One-way repeatedmeasures ANOVA, with time entered as the repeated measure factor,revealed a significant main effect of fentanyl treatment on pawwithdrawal threshold (F(6, 35)=23.42, p<0.0001). Bonferroni's post hoctests revealed significant differences on days 1-3 as compared to day 0.

Both compounds 16 and 33 dose-dependently increased PWT over a doserange of 3.2-32 mg/kg when tested on day 1, as shown in FIG. 6 .Treatment with compound 16 produced a significant main effect asdetermined by one-way repeated measures ANOVA, with treatment entered asthe within subject factor: F(3, 20)=15.10, p<0.0001. Additionally,Bonferroni's post hoc tests revealed significant differences at 10 and32 mg/kg of 16 as compared to the vehicle. Similarly, treatment withcompound 33 produced a significant main effect (F(3, 20)=12.45, p<0.001)and Bonferroni's post hoc tests revealed significant differences at 32mg/kg of 33 as compared to the vehicle.

The foregoing results evidence the effectiveness of compounds 16 and 33to reverse opioid-induced hyperalgesia in rats, as a model system forhuman response to such compounds.

The present disclosure therefore contemplates a neuropeptide FF receptormodulator comprising a compound according to Formula (I) herein, whereinR₂ is selected from —N—(C₂-C₅alkyl)₂ and NH—R₁, wherein R₁ is selectedfrom C₂-C₉ alkyl, heterocyclealkyl, cycloalkylalkyl, aminoalkyl, andarylalkyl; R₃ is selected from C₃-C₉ alkyl, aryl, heteroaryl,heterocycle, heteroarylalkyl, heterocyclealkyl, and arylalkyl; R₄ isselected from H and C₁-C₂ alkyl; and R₅ is selected from C₃-C₉ alkyl,heteroarylalkyl, heteroaryl, heterocyclealkyl, heterocycle,cycloalkylalkyl, and arylalkyl; or a pharmaceutically acceptable saltthereof. Such neuropeptide FF receptor modulator may be constituted withR₂ being NH—R₁, wherein R₁ is selected from C₃-C₉ alkyl,heterocyclealkyl, cycloalkylalkyl, aminoalkyl, and arylalkyl; forexample, R₁ may be C₃-C₆ alkyl, or alternatively, R₁ may be phenethyl,substituted by lower alkoxy, nitro, lower alkyl, halogen or halogenatedlower alkyl.

The neuropeptide FF receptor modulator may be constituted as comprisinga compound of Formula II, wherein R₂ is selected from —N—(C₂-C₅alkyl)₂;and X is S, SO, SO₂, O, NH or CH₂.

Alternatively, the neuropeptide FF receptor modulator may be constitutedas comprising a compound of Formula IIA, wherein R₁ is selected fromC₂-C₉ alkyl, heterocyclealkyl, cycloalkylalkyl, aminoalkyl, andarylalkyl; and X is S, SO, SO₂, O, NH or CH₂; for example, R₁ may beC₃-C₆ alkyl, benzyl or phenethyl, substituted or unsubstituted, and Xmay be oxygen. More specifically, R₁ may be C₃-C₆ alkyl, or R₁ may bephenethyl, substituted by lower alkoxy, nitro, lower alkyl, halogen orhalogenated lower alkyl.

The neuropeptide FF receptor modulator may be constituted as comprisinga compound of Formula III, wherein R₃ is selected from C₃-C₉ alkyl,aryl, heteroaryl, heterocycle, heteroarylalkyl, heterocyclealkyl, andarylalkyl; and R₄ is selected from H and C₁-C₂ alkyl. Such modulator maybe constituted wherein R₃ is benzyl or substituted benzyl, phenethyl orsubstituted phenethyl, and R₄ is H.

Alternatively, such modulator may be constituted wherein R₃ is benzylmono-substituted by methoxy; and R₄ is H. As another alternative, suchmodulator may be constituted wherein R₃ is benzyl or substituted benzyland R₄ is methyl. As a still further alternative, such modulator may beconstituted wherein R₃ is C₃-C₆ alkyl.

The neuropeptide FF receptor modulator may be constituted as comprisinga compound of Formula IV, wherein R₅ is selected from C₃-C₉ alkyl,heteroarylalkyl, heteroaryl, heterocyclealkyl, heterocycle,cycloalkylalkyl, and arylalkyl; and X is S, SO, SO₂, O, NH or CH₂. Suchmodulator may be constituted wherein R₅ is benzyl or substituted benzyl,e.g., wherein R₅ is monosubstituted benzyl and the substituents arehalogen or methoxy at the 2- or 3-position.

The neuropeptide FF receptor modulator in another aspect may beconstituted with the compound having the structure set out in Table 1hereof, wherein R₂ is selected from the group consisting of the R₂species set out in such table.

The neuropeptide FF receptor modulator in a further aspect may beconstituted with the compound having the structure set out in Table 2hereof, wherein R₁ is selected from the group consisting of the R₁species set out in such table.

The neuropeptide FF receptor modulator in an additional aspect may beconstituted with the compound having the structure set out in Table 3hereof, wherein R₃ and R₄ are selected from the group consisting of theR₃ and R₄ species set out in such table.

The neuropeptide FF receptor modulator in yet another aspect may beconstituted with the compound having the structure set out in Table 4hereof, wherein R₅ is selected from the group consisting of the R₅species set out in such table.

The neuropeptide FF receptor modulator in a further aspect may beconstituted with the compound being

The neuropeptide FF receptor modulator in a still further aspect may beconstituted with the compound being

The disclosure further contemplates a pharmaceutical compositioncomprising a neuropeptide FF receptor modulator as variously describedherein, and a pharmaceutically acceptable carrier. The pharmaceuticalcomposition may further comprise an opioid drug, e.g., at least oneselected from the group consisting of fentanyl, morphine, oxycodone,hydrocodone, and buprenorphine. Alternatively, the pharmaceuticalcomposition may further comprise an antipsychotic drug, e.g., at leastone selected from the group consisting of haloperidol and aripiperazole.As a still further alternative, the pharmaceutical composition mayfurther comprise a monoamine reuptake inhibitor, e.g., at least oneselected from the group consisting of fluoxetine and sertraline.

The disclosure also contemplates a method for treating a subject havingor susceptible to a condition or disorder where modulation ofneuropeptide FF receptor activity is of therapeutic benefit, comprisingadministering to the subject having or susceptible to such condition ordisorder a therapeutically effective amount of a neuropeptide FFreceptor modulator as variously described herein. In such method, thecondition or disorder may comprise the use or abuse of one or moreopioid drugs, e.g., fentanyl, morphine, oxycodone, hydrocodone,buprenorphine, heroin, and opioid derivatives of the foregoing. In themethod broadly described above, the therapeutic benefit may comprise atleast partial attenuation of opioid-induced hyperalgesia, e.g., whereinthe opioid-induced hyperalgesia is induced by an opioid drug comprisingat least one selected from the group consisting of fentanyl, morphine,oxycodone, hydrocodone, and buprenorphine.

The method broadly described above may be carried out, wherein theadministering of a neuropeptide FF receptor modulator, constituted asvariously described herein, is performed in a therapeutic interventioncomprising coadministration of a drug for which the neuropeptide FFreceptor modulator attenuates a side effect, e.g., wherein the drug forwhich the neuropeptide FF receptor modulator attenuates a side effect isa drug producing tolerance or hyperalgesia as the side effect.

The method broadly described above may be carried out in specificimplementations, as further comprising administering an effective amountof a second therapeutically effective agent. Alternatively, the methodbroadly described above may be carried out in other specificimplementations, wherein the condition or disorder where modulation ofneuropeptide FF receptor activity is of therapeutic benefit is selectedfrom the group consisting of attenuation of opioid tolerance andattenuation of hyperalgesia.

It therefore will be appreciated that the present disclosurecontemplates a wide variety of compounds, neuropeptide FF receptormodulator agents, pharmaceutical and therapeutic compositions andformulations, and methods of making and using the foregoing.

Accordingly, while the invention has been has been described herein inreference to specific aspects, features and illustrative embodiments ofthe invention, it will be appreciated that the utility of the inventionis not thus limited, but rather extends to and encompasses numerousother variations, modifications and alternative embodiments, as willsuggest themselves to those of ordinary skill in the field of thepresent disclosure, based on the description herein. Correspondingly,the subject matter as hereinafter claimed is intended to be broadlyconstrued and interpreted, as including all such variations,modifications and alternative embodiments, within its spirit and scope.

What is claimed is:
 1. A method of modulating neuropeptide FF receptoractivity in a subject, comprising administering to said subject acompound according to Formula (I):

wherein R₂ is selected from —N—(C₂-C₅alkyl)₂ and NH—R₁, wherein R₁ isselected from C₂-C₉ alkyl, heterocyclealkyl, cycloalkylalkyl,aminoalkyl, and arylalkyl; R₃ is selected from C₃-C₉ alkyl, aryl,heteroaryl, heterocycle, heteroarylalkyl, heterocyclealkyl, andarylalkyl; R₄ is selected from H and C₁-C₂ alkyl; and R₅ is selectedfrom C₃-C₉ alkyl, heteroarylalkyl, heteroaryl, heterocyclealkyl,heterocycle, cycloalkylalkyl, and arylalkyl; or a pharmaceuticallyacceptable salt thereof.
 2. The method of claim 1, wherein R₂ is NH—R₁,wherein R₁ is selected from C₃-C₉ alkyl, heterocyclealkyl,cycloalkylalkyl, aminoalkyl, and arylalkyl.
 3. The method of claim 1,wherein said compound is according to Formula II:

wherein R₂ is selected from —N—(C₂-C₅alkyl)₂; and X is S, SO, SO₂, O, NHor CH₂.
 4. The method of claim 1, wherein said compound is according toFormula IIA:

wherein R₁ is selected from C₂-C₉ alkyl, heterocyclealkyl,cycloalkylalkyl, aminoalkyl, and arylalkyl; and X is S, SO, SO₂, O, NHor CH₂.
 5. The method of claim 1, wherein said compound is according toFormula III:

wherein R₃ is selected from C₃-C₉ alkyl, aryl, heteroaryl, heterocycle,heteroarylalkyl, heterocyclealkyl, and arylalkyl; and R₄ is selectedfrom H and C₁-C₂ alkyl.
 6. The method of claim 1, wherein said compoundis according to Formula IV:

wherein R₅ is selected from C₃-C₉ alkyl, heteroarylalkyl, heteroaryl,heterocyclealkyl, heterocycle, cycloalkylalkyl, and arylalkyl; and X isS, SO, SO₂, O, NH or CH₂.
 7. The method of claim 1, wherein saidcompound has the structure

wherein R₂ is selected from the group consisting of: (1) NHMe; (2) NHEt;(3) NH(n-Pr); (4) NH(n-Bu); (5) NH(s-Bu); (6) NH(t-Bu); (7)NH(n-Pentyl); (8) NH(i-Pentyl); (9) NH(n-hexyl); (10) NH(n-decyl)


8. The method of claim 1, wherein said compound has the structure

wherein R₁ is selected from the group consisting of: (i) Et; (ii)n-pentyl;


9. The method of claim 1, wherein said compound has the structure

wherein R₄ is H, and R₃ is selected from the group consisting of: (a)n-Pr; (b) n-Bu; (c) n-pentyl; (d) n-hexyl;


10. The method of claim 1, wherein said compound has the structure

wherein R₄ is Me, and R₃ is


11. The method of claim 1, wherein said compound has the structure

wherein R₅ is selected from the group consisting of: (I) n-hexyl;


12. The method of claim 1, wherein said compound is


13. The method of claim 1, wherein said compound is


14. The method of claim 1, wherein the compound is a neuropeptide FFreceptor antagonist.
 15. The method of claim 1, wherein the compound isadministered orally, transdermally, parenterally, subcutaneously,intramuscularly, intravenously, intraperitoneally, intracerebrally,intracerebroventricularly, topically, rectally, intranasally,intramuscularly, intravaginally, or via catheter delivery.
 16. Themethod of claim 1, wherein the subject has a condition or disorderrelated to or involving pain management, addiction, opioid tolerance,opioid hyperalgesia, inflammation, feeding, blood pressure, insulinrelease, fever, anxiety, limbic seizure activity, opioid-inducedhypothermia, or cardiovascular modulation.
 17. The method of claim 1,further comprising administration of an opioid, an antipsychotic, or amonoamine reuptake inhibitor to the subject.